Amino acid derivatives and their use as antidepressives and hypotensive agents

ABSTRACT

Derivatives having the general formula:    &lt;IMAGE&gt;  (I)  in which the various radicals, A,B,X,R1, R2, R3 and n have indicated definitions. These compounds have in particular enkephalinase-inhibiting, antalgic, antidepressive, antidiarrhea and hypotensive activities.

This application is a division of application Ser. No. 715,764, filed3/25/85, now U.S. Pat. No. 4,618,708, which is a continuation ofapplication Serial No. 449,687 filed 12/14/82, now abandoned.

The present invention relates to new amino acid derivatives, to theirprocess of preparation and to their therapeutic application.

The derivatives according to the invention are inhibitors of enzymes,and in particular enkephalinase which is an enkephalin-degrading enzyme(B. Malfroy et al, Nature 276, 523 (1978); A. Guyon et al, Life Sciences25, 1605 (1979)) and peptidases, such as aminopeptidases involved in themetabolism of opioid peptides (Hambrook et al, Nature (1976) 262, 782;Pert et al, (1976) Science 194, 330-332; Guyon et al, Biochem. Biophys.Res. Commun. (1979) 88, 919-926.

The compounds capable of inhibiting enkephalinase may thus prolong theeffects of endogenic enkephalins or potentiate the action of syntheticanalogs administered in an exogenous manner. Thus these compounds mayreplace morphinic agents in all their properties without having theserious drawbacks of the latter, particularly in respect ofhabit-forming and dependence phenomena.

Dipeptide derivatives having an enkephalinase-inhibiting action arealready known from the patent application No. FR 80/05 601.

An object of the present invention is to provide new dipeptidederivatives or analogs having in particular this action.

These derivatives according to the invention have the following generalformula: ##STR2## in which

A is a group selected from carbonyl, amino and --CH₂ --CO-- when B=NH;

B is selected from a carbonyl group; an amino group; an amino groupsubstituted with a C₁₋₄ alkyl group, a substituted amino group whosethird valence forms with the R₂ group an alkylenic chain having 2 to 4carbon atoms and optionally containing a sulfur atom or a nitrogen atomand/or optionally substituted with a hydroxy or C₁₋₄ alkoxy group;

R₁ is an hydrogen atom; a straight or branched-chain C₁₋₆ alkyl groupoptionally mono- or polysubstituted with a halogen atom; a cyclohexylgroup; a phenyl or naphthyl group optionally mono- or polysubstitutedwith a halogen atom, a trifluoromethyl or hydroxy group; a thienylgroup; a thiazolyl group; a furyl group; an indolyl group; an imidazolylgroup;

Z is --(CH₂)_(n) -- or ═(CH)--;

n is 0, 1 or 2;

p is 0 when Z is ═CH-- and p is 1

in the other cases;

R₂ is a hydrogen atom; a straight or branched-chain C₁₋₆ alkyl group; aC₁₋₄ alkyl group substituted with a residue α or β thienyl, α or βfuryl, α or β thiazolyl, α or β indolyl, α or β imidazolyl, α or βbenzothienyl, α or β benzimidazolyl, naphthyl or a phenyl groupoptionally mono- or polysubstituted with a halogen atom; a hydroxyalkylgroup; an alkoxyalkyl group optionally substituted on the alkoxy moietywith a phenyl, benzhydryl or pyridyl group optionally N-substituted witha phenylalkyl group whose phenyl nucleus is itself optionallysubstituted with one or more halogen atoms; a phenoxyalkyl group; amercapto alkyl group optionally substituted on the sulfur with astraight or branched-chain alkyl group, phenyl or benzyl group;

R₃ is a hydroxy group; a straight or branched-chain C₁₋₈ alkoxy groupoptionally mono- or polyhalosubstituted with a halogen atom, a hydroxyC₃₋₆ cycloalkyl group, amino or aminoxide group, the latter two radicalsbeing optionally mono- or disubstituted with a C₁₋₄ alkyl group, a C₁₋₈alkoxy group substituted with a thienyl, thiazolyl, pyrrolidinyl,piperidyl, piperazinyl, or morpholinyl group, these latter heterocyclicgroups being optionally substituted with a hydroxy or hydroxy C₁₋₄ alkylgroup; a phenoxy or phenyl C₁₋₄ alkoxy group whose phenyl nucleus isoptionally mono- or poly-substituted with a halogen atom, a hydroxy,C₁₋₄ alcoxy or trihalogenomethyl group; an amino group; an amino groupmono- or disubstituted with a straight or branched-chain C₁₋₈ alkylgroup optionally mono- or polysubstituted with a halogen atom, ahydroxy, mercapto, C₁₋₄ alkylthio, C₁₋₄ alkylsulfinyl radical; an aminogroup optionally mono- or disubstituted with a phenyl or phenyl C₁₋₄alkyl group, the latter two radicals being optionally mono- orpolysubstituted on the phenyl nucleus with a halogen atom; a group ofthe formula ##STR3## in which R₄ and R₅ form with the nitrogen atom towhich they are attached a heterocyclic nucleus having 5 or 6 memberswhich may optionally comprise another heteroatom selected from N, O, Sand this heterocycle being optionally mono- or disubstituted with ahydroxy, hydroxy(C₁₋₄)alkyl, C₁₋₄ alkoxy group, C₁₋₄ alkoxy groupsubstituted with a phenyl radical optionally substituted with a C₁₋₄alkyl radical, hydroxy, C₁₋₄ alkoxy radical or a halogen atom, theheterocycle also optionally comprising as a mono- or disubstituent acarboxy, C₁₋₄ alkoxy carbonyl, C₁₋₄ alkoxy carbonyl group whose alkoxygroup is substituted with a phenyl group optionally substituted with aC₁₋₄ alkyl, hydroxy, C₁₋₄ alcoxy group or a halogen atom;

X is a phosphono group; a substituted phosphonyl group of formula--PO(OR)₂ in which R is a C₁₋₄ alkyl group or an aralkyl group; a sulfogroup; an amino group; a mono-substituted amino group of formula NHR' inwhich R' is an alkyl, cycloalkyl, phenyl, mono- or polyhalophenyl,phenyl C₁₋₄ alkyl, mono- or polyhalophenyl C₁₋₄ alkyl, hydroxy, C₁₋₆alkoxy, aralkoxy, mono- or polyhaloaralkoxy whose alkoxy moiety has from1 to 4 carbon atoms, phosphono group, substituted phosphonyl group offormula --PO(OR)₂ in which R is as defined above or sulfo; adisubstituted amino group of formula ##STR4## in which R" is a hydrogenatom, a C₁₋₆ alkyl, a C₁₋₄ alkoxy carbonyl, aroyl, C₁₋₄ aralkyl groupoptionally mono- or polysubstituted on the aryl nucleus with a radicalselected from a halogen atom, a hydroxy, amino, nitro or trifluoromethylgroup, R"' is a hydrogen atom, an alkyl group optionally mono- orpolysubstituted with a halogen atom or a trifluoromethyl group, a C₁₋₄aralkyl group optionally mono- or polysubstituted on the aryl nucleuswith a radical selected from a halogen atom, a hydroxy, amino, nitro ortrifluoromethyl group; a mono- or disubstituted carbamoyl group offormula ##STR5## in which R" and R"' are as defined above; a C₁₋₄ alkylgroup mono- or polysubstituted with a phosphono group, a disubstitutedphosphono group of formula --PO(OR)₂ in which R is as defined above, ora sulfo group; an aminoalkyl group; a monosubstituted aminoalkyl groupof formula --(CH₂)_(m) --NHR' in which R' is as defined above, and m isan integer of 1 to 4; a disubstituted aminoalkyl group of formula##STR6## in which R", R"' and m are as defined above, a mono- ordisubstituted carbamoyl alkyl group of formula ##STR7## in which R", R"'and m are as defined above; provided that there are not simultaneouslyR₁ =R₂ =X=H, R₃ =OH, n=O, A=CO and B=NH;

and the addition salts thereof with pharmaceutically acceptable acids orbases.

Among the compounds of the formula I defined above, a preferred class ofthe compounds comprises the derivatives in which:

A is a carbonyl, amino, or --CH₂ --CO-- group when B is NH;

B is a carbonyl or amino group or a substituted amino group whose thirdvalence forms with R₂ a C₂₋₄ alkylene radical optionally containinganother heteroatom;

R₁ is a hydrogen atom; C₁₋₄ alkyl group; a phenyl group, optionallymono- or polysubstituted with a halogen atom; an indolyl group;

R₂ is a hydrogen atom; a straight or branched alkyl group optionallysubstituted with an indolyl group;

Z and p are as defined above;

n is 0 or 1;

R₃ is a hydroxy group; C₁₋₄ alkoxy optionally mono- or polysubstitutedwith a halogen atom; a C₁₋₄ alkoxy group substituted with a dialkylaminogroup in which the alkyl groups contains 1-4 carbon atoms,dialkylaminoxide, piperidino or hydroxy piperidino group; a phenyl C₁₋₄alkoxy group whose phenyl group is optionally substituted with a halogenatom; an amino group; C₁₋₆ alkyl amino whose alkyl group is optionallymono- or polysubstituted with a halogen atom; a hydroxy, mercapto, C₁₋₄alkylthio, C₁₋₄ alkylsulfinyl radical; phenyl C₁₋₄ alkyl amino whosephenyl group is optionally mono- or polysubstituted with a halogen atom;pyrrolidino mono- or disubstituted with a hydroxy, carboxy, C₁₋₄alkoxycarbonyl or benzyloxy radical;

X is a phosphono group; a phosphono group substituted with at least abenzyl radical; a sulfo group; an amino group mono- or disubstitutedwith a radical selected from a hydroxy, benzyloxy, phosphono group, or aphosphono group mono- or disubstituted with a benzyl, sulfo, formyl,benzylcarbonyl or mono- or polyhalo C₁₋₄ alkyl carbonyl group; acarbamoyl group mono- or disubstituted with a radical selected from ahydroxy group or a benzyloxy group whose phenyl group is optionallymono- or polysubstituted with a halogen atom, or mono- orpolyhalobenzyl; an alkyl group optionally mono- or polysubstituted witha radical selected from a phosphono group or a phosphono groupdisubstituted with a benzyl, sulfo group, an amino alkyl group mono- ordisubstituted on the nitrogen atom with a radical selected from a C₁₋₄alkyl, hydroxy, benzyloxy, phosphono, sulfo, formyl, acetyl, mono- orpolyhalobenzylcarbonyl, mono- or polyhalo C₁₋₄ alkyl carbonyl group; aC₁₋₄ alkyl group substituted with a carbamoyl radical mono- ordisubstituted with an alkyl, hydroxy, benzyloxy radical whose phenylgroup is optionally mono- or polysubstituted with a halogen atom, mono-or polyhalobenzyl, acetyl or benzoyl.

As a halogen atom, preference is particularly given to fluor.

Unless otherwise indicated, the alkyl groups have preferably 1 to 6carbon atoms and in particular 1 to 4 carbon atoms.

As an aryl group, there is envisaged any mono- or polycyclic aromaticnucleus optionally containing a heteroatom such as nitrogen, sulfur oroxygen, and the phenyl group is particularly preferred.

Specific compounds contemplated by the invention are the followingderivatives (in the following formulae φ=the phenyl group): ##STR8##

Note that the numbers of the derivatives given above do not correspondto the numbers of the examples which comprise the intermediate steps ofsynthesis.

The compounds of formula I have one or two asymmetric carbon atoms. Theytherefore exist in the racemic mixture form or in diastereoisomer forms.All these compounds are within the scope of the present invention. Thesyntheses described below can employ the racemic mixture or one of theenantiomers as the starting substance. When there is employed in theoperational mode of synthesis the racemic starting substance, thestereoisomers obtained may be separated in the product by theconventional chromatographic methods or fractional crystallizationmethods. Generally, the isomer L relative to the carbon atom of theaminoacid constitutes the preferred isomer form.

The compounds of formula I form salts which are also part of thisinvention. The salts comprise the acid addition salts which are formedby reaction with various mineral and organic acids providing acidaddition salts, comprising, for example, the halohydrates (in particularthe hydrochloride and the hydrobromide), sulfate, nitrate, borate,phosphate, oxalate, tartrate, maleate, citrate, acetate, ascorbate,succinate, benzenesulfonate, methanesulfanate, cyclohexane sulfanate,and toluenesulfanate.

The basic addition salts are formed by reaction with bases such as NaOHor by ion exchange reaction.

The salts are formed in a conventional manner by reacting the free formof the product with one or more equivalents of the base or of theappropriate acid providing the desired anion or cation in a solvent or amedium in which the salt is insoluble, or in water and by eliminatingthe water by lyophilization. By neutralizing the salts with an insolubleacid such as a cation exchanging resin in the hydrogen form [for examplethe poly-styreneacid-sulfonic resin Dowex 50® (Mikes, LaboratoryHandbook of Chromatographic Methods (Van Nostrand, 1961), page 256], byeluting with a volatile buffer (for example, pyridine/acetic acid) andextracting with an organic solvent, it is possible to obtain the freeformula and, if desired, it is possible to form another salt.

The compounds of the present invention are prepared by the variousprocesses defined below.

The compounds of formula I may be prepared by a conventional peptidiccondensation reaction between two suitably protected aminoacid residues.

For example, the functional group or groups (ie. the amino, carboxy,hydroxy groups) which are not involved in the reaction forming thepeptide bond (ie. --CONH) in the course of the condensation reaction maybe protected by a protector group or protector groups before thecondensation reaction.

By way of intermediate protector groups of the amino groups there areemployed the usual groups such as t.butoxycarbonyl (Boc),benzyloxycarbonyl (Z), isobornyloxycarbonyl (IBOC) group, etc.).

The carboxylic groups may also be protected, if necessary, byesterification (for example, methyl, ethyl, benzyl esters, etc.).

The condensation reaction is carried out by preferably employing thecoupling of the azides without racemization or the process employingdicyclohexyl carbodiimide/1-hydroxy-benzotriazol termed below DCC/HOBtor DCC/3-hydroxy-4-oxo-3, 4-dihydro-1, 2, 3-benzotriazine (OOBt). By wayof a modification, the activated esters of the fractions may beemployed.

The condensation reaction may be carried out in the presence of asolvent. The solvent may be selected from those known to be of use inpeptide condensation reactions. Thus, there may be mentioned, by way ofexample, the following anhydrous or aqueous solvents: dimethylformamide,dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane,tetrahydrofurane, and the appropriate mixtures of such solvents.

The reaction temperature is chosen within the known range for reactionsforming peptide bonds, for example normally within the range of fromabout -20° C. to about 30° C.

Further, the precursor substances (protected peptides) of the desiredcompounds according to the present invention may also be easily preparedby processes of synthesis in a solid phase.

After the end of the desired condensation reaction, if the product hasprotector groups, they may be eliminated by the usual processes. Amongsuch processes there may be mentioned: the catalytic reaction in thepresence of a catalyst such as palladium black, palladium or carbon,platinum, etc., solvolysis by means of hydrofluoric acid,trifluoroacetic acid, etc. and reduction with metallic sodium in liquidammonia.

There is employed in particular trifluoroacetic acid (TFA) foreliminating the Boc groups (protector amino) and a saponification foreliminating the protector ester groups of the carboxylic groups.

The compounds of formula I in which X=--NH--PO(OH)₂ are obtained inaccordance with Li et al, J. Amer. Chem. Soc. (1954), 77, 1866, by theaction of dibenzylphosphoryl chloride prepared in accordance withAtherton et al, J. Chem. Soc. (1948) 1106, on the suitable dipeptideacid or ester: R'₃ designating the hydrocarbonated fraction correspondsto the ester or acid defined for R₃ ##STR9##

The acid is obtained by deprotection of the benzyl residues uponhydrogenation with Pd/C or PdO.

The phosphonates corresponding to formula I in which X=--PO(OH)₂ areobtained by the action of tris-trimethylsilyl phosphite of formulaP[OSi(CH₃)₃ ]₃ in accordance with Rosenthal et al, Tet. Lett. (1975)977, on a brominated derivative of formula I in which X=Br or CH₂ Br.##STR10##

The brominated compounds in which m=0 are obtained in accordance withthe patent application FR 80/08 601, while the compounds in which m=1are obtained by the addition of HbR on the derivative: ##STR11##

These ethylenic derivatives themselves come from the direct condensationof the suitable substituted acrylic acid with an aminoacid ester:##STR12##

Under mild conditions (20° C.) and in solution in an organic solvent,the peptidic coupling is complete and no addition on the double bond isobserved.

The action of anhydrous hydroxylamine on the brominated derivatives offormula I in which X=Br or --CH₂ Br in accordance with La Noce et al,Ann. Chim. Rome (1968) 58, 393, and Kaminski et al, Roczn, Chem. (1973)47, 653, gives the corresponding hydroxylamino derivative of formula Iin which X=--NHOH or --CH₂ --NHOH.

The action of sulfurous anhydride SO₂ on the preceding hydroxylaminoderivatives gives the corresponding sulfamic acids by adaptation of themethod of Ryer et al. J. Amer. Chem. Soc. (1951), 5675. ##STR13##

The sulfonates corresponding to formula I in which X=--SO₃ H or --CH₂SO₃ H are obtained in accordance with two channels:

1--Direct addition of bisulfite on ethylenic compound in accordance withthe method of Schenck et al, J. Org. Chem. (1951), 16, 1686. ##STR14##

2--Oxidation with oxygen, H₂ O₂ or DMSO (Lowe, J. Org. Chem. (1976) 41,2061) of the corresponding thiols in which X=--SH or --CH₂ SH.

The compounds of formula I in which X=--NH₂ or --NHR' are obtained bythe direct addition of the suitable amines in accordance with the methodof Zilkha, J. Amer. Chem. Soc. (1968) 23, 94.

The compounds of formula I in which ##STR15## are obtained in two steps,by the action of O.benzylhydroxylamine on bromopropionic acid followedby a condensation of the derivative obtained with an ester or an amideof glycine, or directly by the action of O.benzylhydroxylamine on thederivative of formula ##STR16##

The methods employed are described in the following references:

K. Kaminski and T. Sokolowska, Rocz. Chem. 47, (1973), 653-656

T. Kolasa and A. Chimiak, Tetrahedron, 30, (1974), 3591-95.

In a second step, the derivative obtained having the following formula##STR17## is acylated on the NH group by a suitable R"'CO residue inaccordance with the following procedure:

The possible elimination of the residues φ--CH₂ and/or R₃ is effected byhydrogenation and/or treatment with NaOH or TFA (when R₃ =Otbu).

The compounds of formula I in which ##STR18## sont obtained by additionof O.benzylhydroxylamine on an acid of the type ##STR19## or bysubstitution with the same reagent of 3-bromo-2-benzyl propionic acidfollowed by the condensation of the derivative of the following formula:##STR20## obtained in both cases on a suitable ester or an amide ofglycerine.

These derivatives may also be obtained by the direct action ofO.benzylhydroxylamine on a dipeptide of the type ##STR21## obtained asdescribed above.

The derivative obtained of formula ##STR22## may be acylated by themethods described above and the various groupings eliminated by theconventional methods.

Compounds of formula I in which ##STR23## are obtained by condensationof O.benzylhydroxylamine on a suitable malonic ester followed by asaponification.

The compound obtained having formula ##STR24## is condensed inaccordance with the methods of peptidic synthesis on a suitable aminoamide or amino ester so as to obtain the derivative ##STR25##

This compound may be alkylated on the HN-benzyloxy group in accordancewith the conventional methods.

The compounds of formula I in which ##STR26## are obtained for exampleby the action of ethyl α-benzyl mono-succinate of formula ##STR27## of asuitable amino amide or amino ester.

The compound obtained, having the formula: ##STR28## is saponified so asto eliminate the ethyl residue (in the case where R₃ is an ester, thegroup R₃ corresponds to Otbu).

The acid obtained is condensed in accordance with the methods ofpeptidic synthesis with O.benzylhydroxylamine. A derivative having thefollowing formula is obtained: ##STR29## which may be acylated oralkylated on the residue of --O--NH-- by conventional methods.

The compounds in which the bond A--B of the general formula (I) isconstituted by the groups A=NH, B=CO are obtained by Curtius' inversionfrom the suitable acid in accordance with the various methods describedby Mr. Goodman and Mr. Chorey in Perspectives in Peptide Chemistry, Ed.A. Eberle Karger, Basel (1981) pp. 283-294. Among these there isemployed preferably the method with DPPA in accordance with the typicaldiagram: ##STR30##

The compounds in which the bond A--B of the general formula (I) isformed by the groups A=CH₂ CO, B=NH are obtained from ethyl ester of3-carboxy-4-phenyl-3-buteneoic acid (prepared in accordance with Cohenet al (1968) J.A.C.S., 90, 3495) by the following reactions:

The following examples are given by way of illustration of thepreparation of the compounds according to present invention.

EXAMPLE 1 N-(dibenzylphosphoryl) L-phenylalanine-glycine methyl ester(Derivative 1)

2 g of L-phenylalanine-glycine methyl ester hydrochloride (7.4 mmoles)are put in suspension in 20 ml of anhydrous chloroform. 1.50 g oftriethylamine (15 mmoles) are added. The solid enters into solution. Thesolution is cooled to 0° C. and 2.17 g (7.4 mmoles) of dibenzylphosphoryl chloride freshly prepared from dibenzyl phosphite andsulfuryl chloride are added while stirring for 30 minutes. The mixtureis allowed to stand for 20 hours at room temperature. The precipitateformed is filtered and the filtrate is washed in succession with 30 mlof 1N HCl, 20 ml of water, 20 ml of 10% bicarbonate, then 2×30 ml ofwater. The organic solution is dried on anhydrous Na₂ SO₄ and evaporatedunder a vacuum. A pale yellow solid is obtained which is recrystallizedin ethyl acetate.

W 1.8 g; M.P. 106° C. Structure confirmed by NMR. C, H, N analysiscorrect.

EXAMPLE 2 N-phosphoryl L-phenylalanine-glycine methyl ester (Derivative2)

1.5 g of the preceding compound are dissolved in 20 ml of anhydrousmethanol and then hydrogenated at ordinary pressure after addition of0.08 g of palladium oxide. After stirring for 2 hours, the theoreticalquantity of nitrogen has been absorbed. This material is filtered,evaporated to dryness under a vacuum. The pasty residue obtained isrecrystallized in a 50/50 mixture of ether and hexane.

W 0.62 g; M.P. 138°-145° C.

EXAMPLE 3 N-(dibenzylphosphoryl), N'-(2-methoxycarbonyl acetyl)1,1-diamino 2-phenyl ethane (Derivative 3)

1 g of N-(2-methoxycarbonyl acetyl) 1,1-diamino 2-phenyl ethane,obtained as described in the description of the patent FR No. 8 008 601,is treated as in Example 1. After washing of the chloroformic solution,a white solid is obtained by evaporation under a vacuum which iscrystallized in ether.

M.P. 98°-102° C.; W 0.7 g.

EXAMPLE 4 N-phosphoryl, N'-(2-methoxycarbonyl acetyl) 1,1-diamino2-phenyl ethane (Derivative 4)

0.5 of the preceding compound in solution in 10 ml of methanol ishydrogenated as in the Example 2. After crystallization in ether, a greysolid is obtained.

M.P. 168°-172° C.; W 0.21 g.

EXAMPLE 5 N-hydroxy L-phenylalanine-glycine (Derivative 5)

4 g of N-(R, 2-bromo 3-phenyl propanoyl)-glycine (14 mmoles) prepared asdescribed in the patent Fr No. 8 008 601, are dissolved in 100 ml ofanhydrous ethanol. A 0.5 g (15 mmoles) anhydrous solution ofhydroxylamine prepared according to La Noce et al. (1968) Ann. Chim.Rome, 58, 393, in 50 ml of anhydrous ethanol. The mixture is allowed tostand for 48 hours at room temperature, and then evaporated to drynessunder a vacuum. The oily residue is dissolved in 20 ml of water andacidified to pH 1 with concentrated HCl. The acid solution is washedwith 2×30 ml of CHCl₃, then brought to a pH 4 with concentrated NH₄ O4and again extracted with 30 ml of CHCl₃. The organic phases are united,dried and then evaporated to dryness. A glassy solid is obtained whichis purified by crystallization in a 50/50 H₂ O/EtOH mixture.

W 2.8 g; Rf 0.32 in BuOH/AcOH/H₂ O, 4/1/1 (solvent A).

EXAMPLE 6 N-(2-sulfoamino 3-phenyl propanoyl)-glycine (Derivative 6)

1 g of the compound obtained in Example 5 is dissolved in a mixture of30 ml of CHCl₃ and 20 ml of dioxane. A sulfurous anhydride, SO₂, isbubbled through for 5 minutes at 0° C. away from humidity. The gaseouscurrent is stopped and the mixture is allowed to stand at roomtemperature for 30 minutes. A gelatinous precipitate is formed which isfiltered. The filtrate is recrystallized in ethyl acetate.

W 0.168 g; M.P. 237° C.

EXAMPLE 7 N-(2-phosphono 3-phenyl propanoyl)-glycine (Derivative 7)

2 g of N-(R, 2-bromo 3-phenyl propanoyl)-glycine are mixed with, inlarge excess, 11 g (5 equivalents) of tris-trimethyl silyl phosphite,prepared in accordance with Orlov et al. (1970) Chem. Abstr. 72, 21738Y, and heated to 100° C. for 20 hours under a nitrogeneous atmosphere.The oily residue obtained is treated with acetonitrile so as toeliminate the excess reagent. A pale brown wax remains which is stirredfor 12 hours at room temperature with a 50/50 mixture oftetrahydrofuranne (THF) and water. The precipitate formed is collected,dissolved in methanol and then purified by passage on a column of DEAEcellulose (LH20). 0.186 g of a yellow compound is obtained.

M.P. 250° C.

EXAMPLE 8 N-(2-phosphono 3-phenyl propanoyl)-glycine benzyl ester(Derivative 8)

The preceding reaction carried out with 5 g of N-(R, 2-bromo 3-phenylpropanoyl)-glycine benzyl ester under the same conditions (8 equivalentsof the reagent) results in an improved yield of N-(2-phosphono 3-phenylpropanoyl)-glycine benzyl ester.

W 2.10 g; M.P. 208°-228° C.

The latter product may be transformed quantitatively into the precedingproduct by catalytic hydrogenation in the presence of Pd/C in methanol.

EXAMPLE 9 N-(2-sulfo 3-phenyl propanoyl)-glycine (Derivative 9)

3 g (12 mmoles) of N-(S, 2-mercapto 3-phenyl propanoyl)-glycine preparedas described in the patent FR No. 8 008 601, are dissolved in 30 ml ofpure dimethylsulfoxide, 0.43 ml of water (24 mmoles) and 0.3 ml of a 48%solution of HBr are added. The mixture, placed in a three-neck flaskequipped with a distillation head, is slowly brought to the temperatureof 100°-110° C. The dimethylsulfide formed in the course of the reactiondistills at 40°-45° C. When the distillation stops (about 3 hours),there is added 2N NaOH to the liquid residue of the three-neck flaskuntil pH 9. The addition of 100 ml of ethyl acetate results in theprecipitation of 1.4 g of sodium salt. The latter is recrystallized in a50/50 methanol/H₂ O mixture.

W 0.9 g; M.P. 300° C.

This compound may also be obtained with a lower yield by oxidation ofthe N-(S, 2-mercapto 3-phenyl propanoyl)-glycine in accordance with themethod of Vasilevskii et al., Z. Org. Khimil, (1970), 244.

EXAMPLE 10 N-(2-sulfo, 3-phenyl propanoyl)-glycine benzyl ester(Derivative 10)

2 g of N-(S, 2-mercapto 3-phenyl propanoyl)-glycine benzyl esterobtained as described in the patent FR No. 8 008 601, are treated underthe same conditions as before. However, at the end of the reaction, theresidue of the three-neck flask is diluted in 50 ml of water andseparated from the residual DMSO by chromatography on 50-X8 Dowex resinin the acid form. The fractionating is followed by a thinlayer-chromatography (CMC) on a silica gel plate. Eluent BuOH/AcOH/H₂ O,4/1/1, iode developer.

The fractions of Rf=0.61 are united, evaporated under reduced pressure.The wax obtained is crystallized in boiling ethyl acetate.

W 0.42 g; M.P. 260°-280° C.

EXAMPLE 11 N-(2-benzyl propenoyl)-glycine benzyl ester

2.42 g (15 mmoles) of 2-benzyl acrylic acid are dissolved in 30 ml ofTHF and 10 ml of CHCl₃. There is added a solution in 50 ml of THF of1.87 g of glycine benzyl ester hydrochlorate, 2.2 g of triethylamine,then 2.20 g of HOBT and 3.4 g of DCC. The mixture is stirred for 20hours at room temperature. The precipitate of DCU formed is collected.After evaporating to dryness and taking up with ethyl acetate theorganic phase is washed in succession with 2×20 ml of citric acid, then3×20 ml of NaHCO₃. The material is concentrated under a vacuum and therest of the DCU formed is suction filtered. A yellow oil whichcrystallizes slowly is obtained.

W 2.3 g; M.P. 60° C.

EXAMPLE 12 N-(2-benzyl propenoyl)-glycine

2.10 (6.8 mmoles) of N-(2-benzyl propenoyl-glycine benzyl ester,M.P.=60° C., are stirred at 10° C. for 2 hours with a mixture of 30 mlof methanol and 0.27 g of NaOH (6.8 mmoles), dissolved in 5 ml of water.The pH is brought to 1 by the addition of 2N HCl and the material isextracted with ethyl acetate. The solvent is washed with 2×20 ml ofwater, dried and evaporated under a vacuum. 1.36 g of crystals areobtained.

M.P. 82° C., Rf 0.78 in CHCl₃ /MeOH, 9/1.

This compound may be obtained by the condensation of 2-benzyl acrylicacid with glycine methyl ester followed by saponification in accordancewith the following technique. 4.86 g (30 mmoles) of 2-benzyl acrylicacid are dissolved in 50 ml of dry THF. There are added in succession asolution of 3.76 g of glycine methyl ester hydrochlorate (1 equivalent)and 4.2 ml of triethylamine (1 equivalent), then a solution of 4.59 g ofHOBT and 6.8 g of DCC in 40 ml of THF. The mixture is stirred for 18hours at 25° C. The DCU formed is filtered, evaporated to dryness andtaken up with ethyl acetate. It is washed with 2×20 ml of citric acid,then 3× of NaHCO₃ and 2×20 ml of water. The material is dried on Na₂ SO₄and concentrated under a vacuum. The remaining DCU is drained. A paleyollow oil is obtained. W 6.67 g; Rf 0.43 in CHCl₃ /MeOH, 9/1.

6.57 g of N-(2-benzyl propenoyl-glycine methyl ester obtained arestirred for 2 hours at room temperature in a solution of 25 ml of NaOHand 28.2 ml of 1N NaOH.

The methanol is evaporated, 5 ml of water are added, and the mixture isacidified with 2N HCl to pH 1, extracted with ethyl acetate. Afterevaporation, 6.17 g of acid 12 are obtained.

M.P. 82° C.

EXAMPLE 13 N-(2-benzyl propenoyl)-glycine 2-trifluoro ethyl ester

Obtained as in Example 11 by replacing the glycine benzyl ester bytrifluoro ethyl ester obtained by the action of SO₂ Cl on a solution ofglycine in trifluoroethyl alcohol.

Pale yellow oil. Rf 0.62 in CHCl₃ /MeOH.

EXAMPLE 14 N-(2-benzyl propenoyl)-glycine p.fluorobenzyl ester

Obtained as in Example 11 by condensing the 2-benzyl acrylic acid withp.fluorobenzyl ester.

Yellow oil. Rf 0.72.

EXAMPLE 15 N-(3-bromo 2-benzyl propanoyl)-glycine benzyl ester

3 g of N-(2-benzyl propenoyl)-glycine benzyl ester (Example 11) aredissolved in 20 ml of dry CHCl₃. There are added away from humidity andat 0° C. 20 ml of a solution of HBr (3 equivalents), obtained bydissolution of gaseous HBR. The mixture is stirred for 1 hour at 0° C.and then for 10 hours at room temperature. It is concentrated to 10 mlunder a vacuum, and 20 ml of water are added and the mixture isextracted with 2×30 ml of CHCl₃. The organic phase is washer with 20 mlof saturated NaHCO₃ solvent, then with 2×20 ml of water. The material isdried on Na₂ SO₄ and evaporated to dryness. The white solid obtained iscrystallized in ether. W 2.10 g; M.P. 151° C.

EXAMPLES 16 AND 17

The method of Example 15 is applied to the compounds of Examples 13 and14 so as to obtain N-(3-bromo 2-benzyl propanoyl)-glycinetrifluoroethyl, 16, and p.fluorobenzyl, 17, esters.

EXAMPLE 18 N-(3-hydroxyamino, 2-benzyl propanoyl)-glycine benzyl ester(Derivative 11)

1.2 g of the compound of Example 15 is treated as in the Example 5 withanhydrous hydroxylamine. After stirring for 20 hours, the mixture isevaporated under a vacuum. The extraction as in Example 5 provides 0.45g of an amorphous compound.

Rf 0.62 in BuOH/AcOH/H₂ O, 4/1/1.

EXAMPLES 19 AND 20

The method of Example 18 applied to the compounds of Examples 16 and 17respectively provides N-(3-hydroxyamino 2-benzyl propanoyl)-glycine,ethyl trifluoro ester, 19, and N-(3-hydroxyamino 2-benzylpropanoyl)-glycine p.fluorobenzyl ester, 20.

EXAMPLE 21 N-(3-sulfoamino, 2-benzyl propanoyl)-glycine p.fluorobenzylester (Derivative 14)

0.25 g of the compound 20 treated with sulfurous anhydride SO₂, inaccordance with the method described in Example 6, results in thederivative of the following sulfamic acid: N-(3-sulfoamino 2-benzylpropanoyl)-glycine p.fluorobenzyl ester.

W 0.160 g; M.P. 260° C.

EXAMPLE 22 N-(3-phosphono 2-benzyl propanoyl)-glycine benzyl ester(Derivative 15)

0.7 g of N-(3-bromo 2-Benzyl propanoyl)-glycine p.fluorobenzyl ester(example 17), are treated as in Example 7.

0.18 g of N-(3-phosphono 2-benzyl propanoyl)-glycine benzyl ester areobtained.

Yellow product. M.P. 250° C.; Rf 0.32 in BuOH/AcOH/H₂ O, 4/1/1.

EXAMPLE 23 N-(3-sulfo 2-benzyl propanoyl)-glycine benzyl ester(Derivative 16)

4 g (13 mmoles) of N-(2-benzyl propenoyl)-glycine benzyl ester 11 aredissolved in a mixture of 10 ml of CH₃ OH and 50 ml of water containing0.37 g of Na₂ SO₄ (3 mmoles) and 1.04 (10 mmoles) of NaHSO₃. The flaskis then connected to a hydrogenation apparatus operating at ordinarypressure and filled with oxygen. After sweeping, the flask with theoxygen, the reaction is magnetically stirred. After stirring for 20hours, 10.5 ml of oxygen were absorbed. The solution is evaporated undera vacuum to a volume of 10 ml then extracted with 2×20 ml of ethylacetate. The dried solvent is evaporated to dryness under a vacuum. Anamorphous solid is obtained which is recrystallized in methanol. In thisway 1.7 g of the sodium salt of Example 23 are obtained. M.P. 260° C.

This compound may also be obtained by oxidation with DMSO according tothe method described for the compound 10, in starting with N-(3-mercapto2-benzyl propenoyl)-glycine benzyle ester.

EXAMPLE 24 3-ethylamino 2-benzyl propanoyl glycine benzyl ester(Derivative 17)

4 g (13 mmoles) of N-(2-benzyl propenoyl)-glycine p.fluorobenzyl ester,14, are dissolved in 15 ml of pyridine. 1.02 g (13 mmoles) of ethylaminein solution in the pyridine are added. The solution is heated to 120° C.for 2 hours. At the end of the reaction, the solution is concentrated to5 ml, and 50 ml of ether are added. A white product precipitates. It isfiltered, washed with water, and then with ether. It is recrystallizedin a 50/50 CH₃ OH/H₂ O mixture. White crystals are obtained.

W 1.8 g; Rf 0.81 in BuOH/AcOH/H₂ O. 4/1/1.

EXAMPLE 25 N-hydroxy L-alanine-glycine (Derivative 18)

3 g of N-(R, 2-bromo propanoyl)-glycine obtained by condensation of2-bromo propionic acid (prepared according to E. Fischer et al.,Annalen, 357, 1 (1907) with the glycine are dissolved in 100 ml ofanhydrous ethanol and stirred for 48 hours with anhydrous hydroxylamineaccording to Example 5.

0.7 g of a white solid is obtained after crystallization in boilingethanol.

M.P. 260°-270° C.

EXAMPLE 26 N-(2-sulfoamino propanoyl)-glycine (Derivative 19)

A current of SO₂ is bubbled in 0.5 g of the preceding compound dissolvedin a mixture of 10 ml of CHCl₃ and 10 ml of dioxanne. After treatment asin Example 6, 0.130 g of a solid is obtained. M.P. 210°-230° C.

EXAMPLE 27 N-(dibenzylphosphonyl) L-alanine-L-thioproline 2-trifluoroethyl ester (Derivative 20)

4 g of 4-carboxy thiazolidine are dissolved in 20 ml oftrifluoroethanol. 2 ml of SOCl₂ are added and the mixture is refluxedfor 15 mn. After having allowed to stand 20 hours at 25° C. 3.2 g of4-carboxy thiazolidine trifluoro-ethyl ester hydrochlorate are drainedoff.

The coupling between the t.boc L-alanine and the preceding ester iseffected by means of the DCC+HOBT mixture according to conventionaltechniques. The compound obtained is deprotected with the TFA and yieldsafter evaporation and washing with ester 3.7 g of L.Ala-L. thioprolinetrifluoroethyl ester.

2 g of L-Ala-L.thioproline trifluoroethyl ester trifluoroacetate (5.1.mmoles) are put in suspension in 30 ml of dry CHCl₃. There is added at0° C. a mixture of 1.5 g (5.1 mmoles) of freshly prepareddibenzylphophoryl chloride, then 1.02 g of triethylamine in 2 ml of dryCHCl₃. The mixture is stirred for 20 hours at room temperature. Theprecipitate formed is filtered. The organic phase, treated as in Example1, yields a white solid.

Y 2.20 g; M.P. 143°-148° C.

EXAMPLE 28 N-phorphoryl L-alanine-thioproline trifluoroethyl ester(Derivative 21)

1.8 of of the preceding compound are dissolved in 31 ml of dry methanoland hydrogenated at ordinary pressure in the presence of 1 g of 10%Pd/C. After 10 hours, the theoretical quantity (2 equivalents) of H₂were had been absorbed. The material is filtered, evaporated under avacuum and taken up with methanol and chromatographed on cephade×LH 20in CH₃ OH. 1.1 g of a vitrous white solid, soluble in water, isobtained. M.P. 250° C.; Rf 0.82 in BuOH/AcOH/H₂ O, 4/1/1.

EXAMPLE 29 2-bromo-3-phenyl-propanoyl glycine isoamylamide

There are placed in a flask of one liter 11.56 g of 2-bromo-3-phenylpropanoic acid in 50 ml of dry THF. There are added in succession at 5°C., 6.34 g of glycine methyl ester hydrochlorate and 5.1 g oftriethylamine, then 7.73 g of HOBT in 50 ml of THF, and finally 10.42 gof DCC in 50 ml of CHCl₃. The mixture is stirred for 72 hours at 25° C.The DCU formed is filtered and concentrated under a vacuum. The solidresidue is taken up with 100 ml of ethyl acetate. The solution is washedin succession with 50 ml of H₂ O, then with 2×20 ml of 10% of citricacid and finally with 2×20 ml of 10% NaHCO₃. The organic solution isdried on dry Na₂ SO₄ and then evaporated under a vacuum. The crystallineprecipitate is recrystallized with AcOEt. W 11.2 g; M.P. 116° C.; Rdt74%

Analysis C, H, N--NMR in conformity--Rf 0.53 in CHCl₃ /CH₃ OH, 9/1.

2 g of the preceding methyl ester are placed in 20 ml of THF. There areadded in succession at 0° C., 0.61 g of isoamylamine in 10 ml of CHCl₃,then 1.87 g of HOBT, H₂ O, and 1.44 g of DCC in 10 ml of CHCl₃. Themixture is stirred for 12 hours at room temperature. The precipitate ofDCU is drained oof, the solvent evaporated to dryness under a vacuumgives a syrupy residue which is taken up with 2cOEt (50 ml). The organicsolution is washed as before with citric acid, bicarbonate and water.After evaporation to dryness and crystallization in AcOEt, 2.34 g of awhite solid are obtained. M.P. 110° C.; Rdt 94%; NMR correct C, H, N

EXAMPLE 30 N-(2-bensyloxyamino 3-phenyl propanoyl)-glycine isoamylamide(Derivative 22)

1.78 g of the preceding amide are dissolved in 20 ml of MeOH. 1.60 g ofO.benzylhydoxylamine hydrochlorate and 2.59 g of diisopropylethyl amineare then added. The mixture is refluxed for 8 days. The mixture isconcentrated to dryness under a vacuum, and taken up with AcOEt (30 ml),the precipitate of diisopropyl ethylamine salts is filtered. The solventis evaporated under a vacuum and results in an oil (2.61 g) which ispurified on a silica column with the CHCl₃ /CH₃ OH, 98/2 mixture aseluent.

1.04 g of oily product are obtained.

NMR correct C,H,H.

EXAMPLE 31 N-(N-benzyloxy 2-formamido 3-phenylpropanoyl)-glycineisoamylamide (Derivative 23)

0.42 g of the preceding compound is added to 5.2 ml of formic acid(d=1.23). The mixture is cooled to O° C., 0.52 ml of acetic anhydride isadded, and the mixture is stirred for 2 hours at room temperature andconcentrated to dryness under a vacuum. The oil is purified by passageof a silica gel column with MeOH as eluent. The fractions of Rf=0.32 inCHCl₃ /CH₃ OH, 95/5 are united and evaporated to dryness. 0.22 g of apale yellow oil is obtained.

NMR correct C, H, N.

EXAMPLE 32 N-(N-hydroxy 2-formamido 3-phenyl propanoyl)-glycineisoamylamide (Derivative 24)

127 mg of the compound of Example 31 are dissolved in 3 ml of themixture CH₃ OH/AcOH/H₂ O, 4/5/1 and hydrogenated at ordinary pressure inthe presence of 30 mg of 10% Pd/C. After 1 hour, the theoretical volumeis absorbed or filtered, and concentrated to dryness giving a whitesolid. W 88 mg. Rdt 88%; Rf 0.85; BuOH/AcOH/H₂ O, 4/1/1/.

EXAMPLE 33 (a) N-benzyloxy 2-formamido 3-phenyl propanoic acid

0.5 g of N-benzyloxy phenylalanine (obtained by the action ofO.benzylhydroxylamine on 2-bromo 3-phenyl propanoic acid, according toLa Noce et al., Ann. Chim. Rome, 1968, 58, 393), is added to 9.2 ml ofHCO₂ H. 0.5 ml of acetic anyhydride are poured onto the mixture which isstirred for 3 hours at 0° C. It is concentrated to dryness under avacuum. The resinous solid is crystallized in EtOH.

W 500 mg; Rdt 90%; M.P. 186° C.; Rf 0.55 in CHCl₃ /CH₃ OH, 5/1. NMRCorrect C, H, N.

(b) N-(N-benzyloxy 2-formamido 3-phenyl propanoyl glycine benzyl ester(Derivative 25)

0.49 g of the preceding compound are added to 0.552 g of glycine benzylester tosylate in the presence of 0.165 g of triethylamine. There arethen added 0.25 g of HOBT, H₂ O in 10 ml of THF, then 0.69 g ofN-cyclohexyl-N'-[2-(4-morpholinoethyl)] carbodiimide tosylate. Themixture is stirred overnight at room temperature, the DCU formed isfiltered and the solution is concentrated to dryness and taken up withAcOEt. The remaining DCU is filtered and treated as in Example 29. 0.50g of a thick oil is obtained.

Rdt 68%; Rf 0.74 in CHCl₃ /AcOH, 9/1/0.5 NMR correct. Product employedcrude.

EXAMPLE 34 N-(N-hydroxy 2-formamido 3-phenyl propanoyl)-glycine(Derivative 26)

477 mg of the preceding compound are hydrogenated under the conditionsof Example 32, 263 mg of product are obtained.

Rdt 92% of a solid; M.P. 203° C.; NMR correct; C, H, N, O.

EXAMPLE 35 (a) (N-2-benzyloxycarbomoyl 3-phenyl propanoic acid

1.5 g of the ethyl monoester of the ethyl benzylmalonate are coupledwith 1.08 g of O-benzylhydroxylamine, HCl under the conditions of thepeptic synthesis (Example 29). After treatment as for the compound 29,there are obtained 2.17 g (Rdt: 98%) of a pale yellow oil which issaponified without purification with 20 ml of 1N NaOH in 15 ml of EtOHfor 8 hours at 5° C. A slightly insoluble part is filtered, the EtOH isevaporated, and the aqueous phase is extracted with 1N HCl to pH 2-3.The product crystallizes into white paillettes.

M.P. 180° C.; W 1.25 g Rdt 64%; NMR correct, C, H, N.

(b) N-[2-(N-benzyloxy carbamoyl) 3-phenyl propanoyl]-glycine benzylester (Derivative 27)

0.5 g of the compound obtained at (a) is coupled with 0.56 g of glycinebenzyl ester according to the conditions of Example 33b. Aftertreatment, 0.56 g of product is obtained.

Rdt 75% of an amorphous white solid; M.P. 70° C.; NMR; C, H, N.

EXAMPLE 36 N-[2(N-hydroxy carbamoyl) 3-phenyl propanoyl]-glycine(Derivative 28)

0.5 g of the compound 35 are hydrogenated under the conditions ofExample 32. 274 mg of product are obtained M.P. 183° C.; RF 0.47 in A.

EXAMPLE 37 N-[2-(N-benzyloxy carbamoyl) 3-phenyl propanoyl]-glycinemethyl ester (Derivative 29)

0.6 g of the compound 35a is combined in accordance with the usualmethod (Example 33b) with 0.25 g of glycine methyl ester hydrochloride.After conventional treatment (Example 33b) 0.74 g of a white solid isobtained.

M.P. 107° C. (EtOH); Rdt 64%; Rf 0.7 in CHCl₃ /CH₃ OH/AcOH, 0/1/0.5; NMRcorrect; C, H, N.

EXAMPLE 38 N-[2-(N-hydroxy carbamoyl) 3-phenyl propanoyl]-glycine methylester (Derivative 30)

0.4 g of the compound 37 is hydrogenated at ordinary pressure under theconditions of Example 32. 242 mg of a white solid are obtained.

M.P 174° C.; Rdt 80%; Rf 0.44 in (B) CHCl₃ /CH₃ OH/AcOH, 0/1/0.5.

EXAMPLE 39 N-[2-(N-benzyloxy carbamoyl) 3-phenyl propanoyl]-glycine(Derivative 31)

0.368 g of the compound 35a are combined with 0.32 g of glycinebenzylamide trifluoroacetate under the conditions of Example 33b. 0.47 gof a white solid is obtained.

M.P. 140° C.; Rdt 88%; Rf 0.63 in (B); NMR correct; C, H, N

EXAMPLE 40 N-[2-(N-hydroxy carbamoyl 3-phenyl propanoyl]-glycine(Derivative 32)

0.25 g of the preceding compound are hydrogenated under the conditionsof Example 32. 0.20 g of a white solid is obtained.

M.P. 166° C.; Rdt 87%; Rf 0.84 in (A); NMR correct; C, H, N

EXAMPLE 41 N-[2-(N-benzyloxy carbamoyl) 3-phenyl propanoyl]-glycineisoamylamide (Derivative 33)

0.7 g of the compound 35a are combined with 0.65 g of glycineisoamylamide trifluoroacetate under the conditions of Example 34. 0.94 gof white crystals is obtained.

M.P. 143° C.; Rdt 88%; Rf 0.95 in (A) NMR correct; C, H, N

EXAMPLE 42 N-[2-(N-hydroxy carbamoyl) 3-phenyl propanoyl]-glycineisoamylamide (Derivative 34)

0.4 of the preceding compound is hydrogenated under the conditions ofExample 32. 0.30 g of white crystals is obtained.

M.P. 186° C.; Rdt 90%; Rf 0.84 in (B)

EXAMPLE 43 (a) 3-benzyloxyamino 2-benzyl propanoic acid

5.07 g of triethylamine is added at 0° C. while stirring to a solutionof 7.87 g of O.benzylhydroxylamine hydroxide in a mixture of 50 ml ofwater and 50 if of CHCl₃. The organic phase is separated out, washedwith 3×50 ml of water, dried and evaporated under a vacuum. 6 g ofO.benzylhydroxylamine base are obtained. The latter is added away fromair in solution in 20 ml of MeOH to a solution of 2 g of 2-benzylacrylic acid in 10 ml of MeOH. The mixture is refluxed for 7 days andconcentrated under a vacuum. The oil obtained is dissolved in 100 ml ofAcOEt and N NaOH is added until a pH 10 is reached. The aqueous phase isdecanted and acidified with N HCl until a pH 1-2 is reached. The acidphase is extracted with AcOEt (2×50 ml), dried and evaporated under avacuum. The oil obtained slowly crystallizes.

M.P. 40° C.; W 1.6 g; Rdt 46%; Rf 0.47 in CHCl₃ /MeOH, 9/1; NMR correct;C, H, N.

(B) 3-(N-benzyloxy formamido)2-benzyl propanoic acid

1.08 g of the preceding compound are added to 19 ml of HCO₂ H (d=1.23).1.9 ml of acetic anhydride are poured at 0° C. while stirring. Themixture is stirred for 5 hours at 0° C. and concentrated to drynessunder a vacuum. The thick oil is triturated in n-hexane. A solid isobtained.

M.P. <40° C.; Rf 0.6 in CHCl₃ /CH₃ OH, 9/1; W 1.2 g; Rdt 100%; NMRcorrect.

The compound is employed without subsequent purification for thecouplings.

(c) N-[N-benzyloxy 3-formamido 2-benzyl propanoyl]-glycine isoamylamide(Derivative 35)

0.15 g of the compound obtained at (b) in solution in 15 ml of dry THFis added to 0.12 g of glycine isoamylamide trifluoroacetate and 0.048 gof triethylamine in 20 ml of THF. There are added in succession to themixture 73 mg of HOBT in 5 ml of THF, then 0.2 g of DCC (tosylate) in mlof THF. The solution obtained is stirred for 12 hours at roomtemperature. The mixture is treated as in Example 29. The oil obtained(98 mg) is chromatographed on a silicon gel column (eluent CH₃ OH/CHCl₃2/98). 64 mg of oil are obtained.

RF 0.28 in CHCl₃ /MeOH, 9/1; Rdt 30%; NMR correct.

EXAMPLE 44 N-(N-hydroxy 3-formamido 2-benzyl propanoyl)-glycineisoamylamide (Derivative 36)

60 mg of the preceding compound are hydrogenated as in Example 32. 33 mgof a white solid are obtained.

Rdt 70%; Rf 0.52 CHCl₃ /MeOH, 9/1; NMR correct; C, H, N.

EXAMPLE 45 N-(N-benzyloxy 3-formamido 2-benzyl propanoyl)-glycine benzylester (Derivative 37)

0.15 g of the derivative 43b in solution in 20 ml of dry THF are reactedwith 0.16 g of glycine benzyl ester tosylate under the conditionssimilar to Example 45c. After successive washings, 0.21 g of a thick oilis obtained.

Rdt 97%; RF 0.7 (b); NMR correct; C, H, N.

EXAMPLE 46 N-(N-hydroxy 3-formamido 2-benzyl propanoyl)-glycine(Derivative 38)

0.19 g of the preceding compound is hydrogenated at ordinary pressureunder the conditions of Example 32. 103 mg of white solid are obtained.

M.P. 182° C.; Rf 0.52 in (A)

EXAMPLE 47 N-(N-benzyloxy 3-formamido 2-benzyl propnaoyl)-glycine methylester (Derivative 39)

0.6 g of the compound 43b is reacted under the conditions of thepeptidic coupling (Example 29) with 0.24 g of the glycine methyl ester(hydrochloride). After the usual treatments, 0.36 g of an oil isobtained.

Rdt 50%; Rf 0.77 in CHCl₃ /CH₃ OH, R/1.

The compound is employed without subsequent purification for preparingthe derivative 48.

EXAMPLE 48 N-(N-hydroxy 3-formamido 2-benzyl propanoyl)-glycine methylester (Derivative 40)

0.30 g of the derivative 47 is hydrogenated at ordinary pressure as inExample 32. 226 mg of a white solid are obtained. RDT 98%; Rf 0.51 inCHCl₃ /CH₃ OH, 5/1; NMR correct; C, H, N.

EXAMPLE 49 (a) 3-(N-benzyloxy acetamido)2-benzyl propanoic acid

0.4 g of the compound 43a is dissolved in 7 ml of pyridine. 0.7 ml ofacetic anhydride is added at 0° C. After stirring for 5 hours at roomtemperature, the mixture is concentrated to dryness under a vacuum andtriturated with ethyl ether. After decantation, 0.412 g of a thick oilis obtained.

Rf 0.69 in CHCl₃ /CH₃ OH, 5/1; Rdt 93%; NMR correct; C, H, N.

(b) N-(N-benzyloxy 3-acetamido 2-benzyl propanoyl)-glycine benzyl ester(Derivative 41)

0.52 g of the preceding compound is dissolved in 20 ml of dry THF andtreated under the conditions of the peptidic coupling with 0.56 g ofglycine benzyl ester tosylate. After treatment as in Example 29, 0.42 gof a lac is obtained.

Rf 0.81 in CHCl₃ /CH₃ OH, 5/1; Rdt 53%; NMR correct; C, H, N.

EXAMPLE 50 N-(N-hydroxy 3-acetamido 2-benzyl propanoyl)-glycine(Derivative 42)

0.2 g of the preceding compound is hydrogenated at ordinary pressure asin Example 32. 120 mg of a white solid are obtained.

Rdt 100%; M.P. 176° C.; Rf 0.5 in (A); NMR correct; C, H, N.

EXAMPLE 51 N-(N-benzyloxy 3-acetamido 2-benzyl propanoyl)-glycinebenzylamide (Derivative 43)

0.4 g of the compound 49a is coupled under the conditions of Example 29with 0.35 g of benzylamide glycine trifluoroacetate. After the usualtreatments 0.28 g of a solid is obtained.

M.P. 82° C.; Rdt 46%; RF 0.73 in CHCl₃ CH₃ OH, 5/1; NMR correct; C. H.N.

EXAMPLE 52 N-(N-hydroxy 3-acetamido 2-benzyl propanoyl)-glycinebenzylamide

0.2 g of the preceding compound is hydrogenated at ordinary pressureunder the conditions of Example 32. After treatment, 136 mg of whitecrystals are obtained.

M.P. 147° C.; Rdt 84%; Rf 0.58 in CHCl₃ /CH₃ OH; NMR correct; C. H. N.

EXAMPLE 53 N-(3-ethoxycarbonyl 2-benzyl propanoyl)-glycine tertiobutylester

7 g of 3-ethoxycarbonyl 2-benzyl propanoic acid (0.03 mole) prepared inaccordance with S. G. COHEN (U.A.C.S., 90, 3495-3502, 1968) aredissolved in a mixture of 30 ml of dry THF and 10 ml of anhydrous DMF.There are added in succession while stirring at 0° C. 7.5 g of glycinetertiobutylic ester trifluoroacetate and 3.1 g of triethylamine in 10mlg of THF, then 4.02 g of HOBT and 5.40 g of DCC in 20 ml of THF. Themixture is stirred for 12 hours at room temperature and the DCU formedis filtered and concentrated to dryness under a vacuum. A syrupy residueis obtained which is taken up with 100 ml of AcOET. A new precipitate ofDCU is filtered and the organic solution is washed with 10% of citricacid (3×20 ml), 10% NaHCO₃ (3×20 ml) and then once with 30 ml of water.The solution is dried and evaporated under a vacuum and a pale yellowoil is obtained.

Rf 0.61 in (B); W 8.1. g; Rdt 82%; NMR correct; C, H, N.

EXAMPLE 54 N-(3-carboxy 2-benzyl propanoyl)-glycine tertiobutyl ester

5 g of the preceding compound are dissolved in 10 ml of the mixture, 5ml of CH₃ OH, 20 ml of water. 1 ml of NaOH is added and the mixture isstirred for 4 hours at room temperature. A slightly insoluble part isfiltered off and extracted once with 50 ml of ether. The alkalyneaqueous phase is acidified to pH 1-2 with N HCl and extracted with 3×20ml of AcOEt. The organic phases are combined, washed with water (2×20ml) then dried and evaporated to dryness under a vacuum. A white solidis obtained which is recrystallized in MeOH/EtOH, 20/80.

W 4.0 g; Rdt 87%; M.P. 92°-95° C.; Rf 0.76 (A) NMR correct; C, H, N.

EXAMPLE 55 N-(N-benzyloxy 3-carbamoyl 2-benzyl propanoyl)-glycinetertiobutyl ester (Derivative 45)

2.0 g (6.5 mmoles) of the preceding derivative are dissolved in 20 ml ofTHF and 50 ml of DMF. 1.0 g of O.benzylhydroxylamine hydrochloride in 10ml of CHCl₃ is added, then there are added in succession 0.65 g of oftriethylamine, 1 g of HOBT, H₂ O and 1.25 g of DCC in solution in 10 mlof CHCl₃. The mixture is stirred for 12 hours at room temperature, theDCU is filtered and evaporated under a vacuum to dryness and taken upwith 50 ml of AcOEt and the washings are carried out as in Example 29.In this way 2.1 of a colourless oil are obtained.

Rdt 80%; Rf 0.38 in (B);

The oil is employed without subsequent purification for the followingsteps. NMR correct.

EXAMPLE 56 N-(N-benzyloxy 3-carbamoyl 2-benzyl propanoyl)-glycine(Derivative 46)

0.5 g of the preceding compound are dissolved in 2 ml of TFA in thepresence of 0.1 ml of anisole. The mixture is stirred at 0° C. for 30mn, and then for 1 hour at room temperature. It is then evaporated todryness under a vacuum. The oil obtained is triturated with 3×10 ml ofdry ether. A gummy precipitate appears after with 5×10 ml of dry ether.The powder obtained is crystallized in EtOH. White crystals.

M.P. 176° C.; W 0.21 g; Rdt 51%; NMR correct; C, H, N.

EXAMPLE 57 N-(N-hydroxy 3-carbamoyl 2-benzyl propanoyl)-glycine(Derivative 47)

0.15 g of the preceding compound are hydrogenated at ordinary pressureunder the conditions of Example 32. 0.11 g of a crystallized white solidis obtained.

M.P. 252° C.; Rdt 100%; Rf 0.61 in (A) NMR correct; C, H, N.

EXAMPLE 58 N-(N-benzyloxy 3-carbamoyl 2-benzyl propanoyl)-glycinebenzylamide (Derivative 48)

1 g of 3-ethoxycarbonyl 2-benzyl propanoic acid is dissolved in amixture of 30 ml of dry THF and 10 ml of anhydrous DMF. 1.2 g of glycinebenzylamide trifluoroacetate are added without stirring. The coupling iseffected under the conditions of Example 29. After the usual washings,1.5 g of an oil which slowly crystallizes are obtained.

M.P. 62° C.; W 0.92 g; Rdt 76%; Rf 0.52 in (B) NMR correct; C, H. N.

EXAMPLE 59 N-(N-hydroxy 3-carbamoyl 2-benzyl propanoyl)-glycinebenzylamide (Derivative 49)

0.3 g of the preceding compound are hydrogenated at ordinary pressureunder the conditions of Example 32. 0.12 g of a white solid is obtained.

M.P. 207° C.; W 0.12; Rdt 50%; NMR correct; C, H, N.

EXAMPLE 60 (a) 2-(N-benzyloxy benzylcarboxamido)3-phenyl propanoic acid

2 g of N-benzyloxy phenylalanine (KOLASA, tetrahedron, 30, 3591-3595,1974) are placed in a mixture of 10 ml of water and 10 ml of NaOH. 1.0 gof phenylacetyl chloride is added at 0° C. The mixture is extracted oncewith 20 ml of ether and then the aqueous phase is acidified to pH 2 with2N HCl, and extracted with 3×10 ml of ether, dried and evaporated undera vacuum. 1.7 g of crystals are obtained.

M.P. 112° C.; Rdt 72%; Rf 0.42 (B); NMR correct; C, H, N.

(b) N-[2-(N-benzyloxy benzylcarboxamido)3-phenyl propanoyl]-glycinebenzyl ester (derivative 50)

1 g of the preceding comound is dissolved in 20 ml of dry THF. A mixturein 10 ml of THF and 10 ml of CHCl₃ of 0.87 g of glycine benzyl estertosylate and 0.27 g of triethylamine is added. After stirring for 5 mnat 0° C., there are added in succession at 0° C. 1 ml of HOBT, H₂ O and1 ml of DCC under the conditions of the peptidic coupling (Example 29).After 12 hours the usual treatment is effected. 1.3 g of a colourlessoil purified by chromatography on a silica column (CHCl₃ /CH₃ OH, 80/20)are obtained.

Rdt 91%; Rf 0.65 in the same solvent. NMR correct; C, H, N.

EXAMPLE 61 N-[2-(N-hydroxy benzylcarboxamido)3-phenyl propanoyl]glycine(derivative 51)

1 g of the preceding compound is hydrogenated at ordinary pressure as inExample 32. 0.42 g of white crystals (EtOH) is obtained.

Rdt 62%; M.P. 125° C.; Rf 0.34 in (B) NMR correct; C, H, N.

EXAMPLE 62 (a) 2-(N-benzyloxy β-trifluoroethyl carboxamido)-3-phenylpropanoic acid

2 g of N-benzyloxy phenylalanine are treated as in Example 60a with 1.10g of trifluoromethyl acetic acid chloride. 1.5 g of oil which slowlycrystallizes are obtained.

M.P. 82° C.; Rdt 53%; Rf 0.21 in (B); NMR correct; C, H, N.

(b) N-2-[(N-benzyloxy β-trifluoroethyl caboxamido)3-phenylpropanoyl]-glycine benzyl ester (Derivative 52)

1 g of the preceding compound is coupled under the conditions of thepeptidic coupling (Example 29) with 0.9 g of glycine benzyl estertosylate. After the usual treatments, 1.1 g of a pasty solid areobtained which solid is used without prior purification for thefollowing experiment.

Rf 0.42 in (B).

EXAMPLE 63 N-[2-(N-hydroxy β-trifluoroethyl carboxamido)3-phenylpropanoyl]-glycine (Derivative 53)

0.5 g of the preceding compound is hydrogenated under the usualconditions (Example 32). 0.25 g of a white solid is obtained.

M.P. 208° C.; Rdt 83%; Rf 0.64 in (A).

EXAMPLE 64 N-[2-(N-benzyloxy N-p.fluorobenzyl carbamoyl)3-phenylpropanoyl]-glycine benzyl ester (Derivative 54)

0.5 of the compound 35b is dissolved in 30 ml of dry THF. An equivalent(0.084 g) of freshly prepared of sodium ethylate if added. The mixtureis stirred for 10 hours at room temperature under a nitrogeneouscurrent, and then there is slowly added 0.14 g of p.fluorobenzylchloride and the mixture is allowed to stand for 10 hours at roomtemperature and then refluxed for 2 hours. The material is concentratedto dryness, taken up with AcOEt, washed with water, dried, evaporatedunder a vacuum. 0.12 g of a colourless oil is obtained which is purifiedby passage on a silica gel column (eluent CHCl₃ /Et₂ O, 50/50).

Rf 0.75 in (B).

EXAMPLE 65 N-[2-(N-hydroxy N-p.fluorobenzyl carbamoyl)3-phenylpropanoyl]-glycine (Derivative 55)

0.08 g of the preceding compound is hydrogenated at ordinary pressure asin Example 32. 0.035 g of a white solid is obtained.

M.P. 164° C.; Rf 0.12 in (A); NMYR correct; C, H, N.

EXAMPLE 66 N-[N-benzyloxy N-p.fluorobenzyl 3-carbamoyl 2-benzylpropanoyl]-glycine tertiobutylic ester (Derivative 56)

0.5 g of the compound 55 is treated as in Example 64 with p.fluorobenzylchloride. After treatment, there is obtained 0.42 g of an oil (Rf=0.55in (B)) employed without subsequent purification for preparing thecompound 74.

EXAMPLE 67 N-[3-(N-benzyloxy N-p.fluorobenzyl carbamoyl)2-benzylpropanoyl]-glycine (Derivative 57)

0.42 of the preceding compound is dissolved in 1 ml of TFA at 0° C. Thesolution is left to stand without stirring for 30 mn, and evaporatedunder a vacuum. The residual oil is washed with 10×10 ml of dry ether toa neutral ph. A hygroscopic white powder is obtained.

Rf 0.75 in (B); W 0.30 g; Rdt 80%; NMR correct.

EXAMPLE 68 N-[3-(N-hydroxy N-p.fluorobenzyl carbamoyl)2-benzylpropanoyl]-glycine (Derivative 58)

0.3 g of the preceding compound is hydrogenated as in Example 32. 0.15 gof a crystallized solid is obtained.

M.P. 212° C.; Rdt 62%; NMR correct; C, H, N.

EXAMPLE 69 (a) 3-(N-benzyloxy p.fluorobenzylcarboxamido)2-benzylpropanoic acid

5 g of the compound 43a are condensed with 3.05 ml of p.fluorophenylacetyl chloride as in Example 60. After the usual treatment 3.8 g ofpale yellow crystals are obtained.

M.P. 96° C.; Rdt 51%; NMR correct; C, H, N.

(b) N-[3-(N-benzyloxy p.fluorobenzylcarboxamido)2-benzylpropanoyl]-glycine (Derivative 59)

3 g of the preceding compound are condensed as in Example 29 with 2.32 gof glycine benzyl ester tosylate. 3.52 g of oil are obtained, which oilis employed without purification for preparing the compound 70.

EXAMPLE 70 N-[3-(N-hydroxy p.fluorobenzylcarboxamido)2-benzylpropanoyl]-glycine (Derivative 60)

3 g of the compound 69b are hydrogenated at ordinary pressure under theconditions of Example 32. After crystallization in EtOH, 1.10 g of awhite solid are obtained.

M.P. 174° C.; Rdt 54%; Rf 0.32 in (A) NMR correct; C, H, N.

EXAMPLE 71 (a) 3-(N-benzyloxy β-trifluoroethylcarboxamido)2-benzylpropanoic acid

3 g of the compound 43a are condensed with 1.54 g of trifluoromethylacetic acid chloride as in Example 60. 3.12 g of yellow crystals areobtained.

M.P. 64° C.; Rdt 76%; Rf 0.45 in (B) NMR correct; C, H, N.

(b) N-[3-(N-benzyloxy trifluoromethyl acetamido)2-benzylpropanoyl]-glycine benzyl ester (Derivative 61)

2 g of the preceding compound are condensed as in Example 29 with 1.7 gof glycine benzyl ester tosylate. There are obtained 1.62 g of a yellowoil which is employed without subsequent purification for thepreparation of compound 72.

EXAMPLE 72 N-[3-(N-hydroxy trifluoromethyl acetamido)2-benzylpropanoyl]-glycine (Derivative 62)

1 g of the compound 71 is hydrogenated at ordinary pressure under theconditions of Example 32. After crystallization in AcOEt, 0.62 g of apale yellow solid is obtained.

M.P. 131° C.; Rdt 92%; Rf 0.44 in (A) NMR correct; C, H, N.

EXAMPLE 73 N-(N-hydroxy 3-formamido 2-benzyl propanoyl)-glycine p-fluorobenzylamide (Derivative 63)

0.6 g of the compound 45b is reacted under the conditions of thepeptidic coupling (Example 29) with 0.4 g of glycine para-fluoro benzylamide. After the usual treatments, 0.46 g of an oil is obtained.

Rdt 50%; Rf 0.72 in CHCl₃ /CH₃ OH, 5/1.

The compound is then hydrogenated at ordinary pressure as in Example 32.360 mg (Rdt 95%) of a white solid are obtained.

M.P. 175° C.; Rf 0.53 in CHCl₃ /CH₃ OH, R/1 NMR correct; C, H, N.

EXAMPLE 74 N-(N-hydroxy 3-formamido 2-benzyl propanoyl)-tryptophanep-fluoro benzylamide (Derivative 64)

0.6 g of the compound 45b is reacted under the conditions of thepeptidic coupling (Example 29) with 0.6 g of tryptophane para-fluorobenzyl amide. After the usual treatments, 0.54 g of an oil is obtained.

Rdt 50%; Rf 0.7 in CHCl₃ /CH₃ /OH, R/1.

0.50 g of this compound are employed without subsequent purification fora hydrogenation at ordinary pressure, as in Example 32. 395 mg of awhite solid are obtained.

Rdt 95%; Rf 0.52 in ChCl₃ /CH₃ OH, R/1; NMR correct; C, H, N.

EXAMPLE 75 N-(N-hydroxy 3-formamido 2-benzyl propanoyl)-alanine p-fluorobenzylamide (Derivative 65)

0.600 g of the compound 45b are reacted under the conditions of thepeptidic coupling (Example 29) with 0.20 g of alanine p-fluoro benzylamide. After the usual treatments 0.38 g of an oil is obtained.

RDT 55%; Rf 0.79 in CHCl₃ /CH₃ OH, 5/1.

0.35 g of this compound are employed without subsequent purification forhydrogenation at ordinary pressure, as in Example 32. 220 mg of a whitesolid are obtained. RF 0.58 in CHCl₃ /CH₃ OH, R/1; NMR correct; C, H, N

EXAMPLE 76 [2-(N-hydroxy carbamoyl) 3-phenyl propanoyl]-leucine p-fluorobenzylamide (Derivative 66)

0.600 g of the compound 35a are coupled with 0.50 g of leucine p-fluorobenzyl amide under the conditions of Example 34. After treatment, 730 mgof an amorphous white solid (Rdt 75%) are obtained.

0.700 g of this compound is hydrogenated under the conditions of Example32. 520 mg of an unstable white solid compound are obtained.

Rdt 90%; M.P. 175° C.; Rf 0.48.

EXAMPLE 77 N-(N-hydroxy 2-formamido 3-pentafluorophenylpropanoyl)-glycine butyl ester (Derivative 67)

9 g of pentafluorobenzaldehyde are condensed on 7 g of diethyl malonatein accordance with the process described in Organic Synthesis vol. III,page 377. 10 g (69%) of diethyl pentafluorobenzylmalonate are obtainedwhich is hydrogenated at ordinary pressure in the presence of palladiumon charcoal into diethyl pentafluorobenzylmalonate (92%).

By adopting the process described in the patent FR 8 008 601 forpreparing the benzylacrylic acid, the pentafluorobenzyl acrylic acid isobtained. The latter compound gives by means of the processes describedin Examples 45 and 46 3-(N-benzyloxyformamido 2-pentafluorobenzylpropanoic acid. By condensing the latter compound with glycine butylester in accordance with the process of Example 49 and catalytichydrogenation under the conditions of Example 32, the derivative 77 isobtained.

White solid--RF 0.53, CHCl₃ /CH₃ OH, R/l; M.P. 55° C. NMR correct; C, H,N.

EXAMPLE 78 (a) N-benzyloxy 2-formamido 1-benzyl ethylamine

1 g of the compound 43b is treated in accordance with the process ofNinomiva et al (Chem. Pharm. Bull. 22, 1398 (1974) withdiphenylphosphorazide in benzene in the presence of triethylamne, thenwith tertiobutyl alcohol so as to give 610 mg (50%) of a pale yellowoily compound.

The oil is treated with trifluoroacetic acid and yields 540 mg oftrifluoroacetate of 78a (85%). White solid.

M.P. 125° C.; Rf 4/1/1 0.42.

(b) [N-formyl N-benzyloxy N'(2)benzyloxycarbonyl)acetyl] 1,2-diamino2-benzyl ethane (Derivative 68)

500 mg of trifluoroacetate 78a are coupled with 245 mg of malonic acidmonobenzylester in the presence of DCC/HOBT in accordance with theconventional process of peptidic coupling (Example 43c). Aftersuccessive washings, 500 mg of a thick colourless oil are obtained.

Rdt 90%; Rf 0.55 in CHCl₃ /MeOH, 5.1.

EXAMPLE 79 [N-formyl N-hydroxy N'(2-carboxy)actyl] 1,2-diamino 2-benzylethane (Derivative 69)

300 mg of the preceding compound are hydrogenated at ordinary pressureunder the conditions of Example 32. 160 mg of a white solid areobtained.

M.P. 182° C. (90%); Rf. 0.20 in ChCl₃ /MeOH/acetic acid, 9/1/0.5.

EXAMPLE 80 [N-formyl β-benzyloxy N'(2-benzylcarboxamido)acetyl]1,2-diamino 2-benzyl ethane (Derivative 70)

500 mg of trifluoroacetate of 78a are coupled with 245 mg of malonicacid monobenzylamide in the presence of DCC/HOBT in accordance with theprocess of Example 43c.

After successive washings, 510 mg of a thick colourless oil which slowlycrystallizes are obtained.

Rdt 91%; Rf 0.40 in CHCl₃ /MeOH/acetic acid 9/1/0.5.

EXAMPLE 81 [N-formyl N-hydroxy N'(2-benzylcarboxamido)acetyl]1,2-diamino 2-benzyl ethane (Derivative 71)

300 mg of the preceding compound are hydrogenated at ordinary pressureunder the conditions of Example 32. 215 mg of a white solid (90%) areobtained.

M.P. 145° C.; Rf 0.30 in CHCl₃ /MeOH/acetic acid 9/1/0.5.

The pale yellow oil obtained crystallizes in plates.

M.P. 101°-108° C.; W 1.6 g; Rf 0.40 in CHCl₃ /MeOH, 7/3; C, H, N.

(b) 3-(N-benzyloxy formamido) 2-(β-indolylmethyl)propanoic acid

(1) Preparation of 3-benzyloxyamino 2-β-indolylmethyl propanoic acid.

There is added at room temperature a solution of 1.12 g (0.009 mole) ofO.benzyl hydroxylamine in solution in 20 ml of CH₃ OH, to 1.5 g (0.008mole) of the compound of Example 82a-2 dissolved in 20 ml of CH₃ OH. Themixture is refluxed for 5 hours. The treatment is then as in Example 43.The oil obtained crystallizes.

M.P. 67° C.; W 1.4 g; Rf 0.23 in CHCl₃ /MeOH, 9/1. NMR correct.

(2) 1.3 g of the preceding compound are added to 17 ml of HCO₂ H andthen the mixture is treated and extracted under the conditons of Example35. A thick oil which does not crystallize is obtained.

Rf 0.4 in CHCl₃ /CH₃ OH, 9/1; W 0.7 g. The NMR being correct, theproduct is employed without purification for the following reactions.

(c) N-[3-(N-benzyloxy formamido 2-β-indolylmethyl propanoyl] glycinebenzyl ester (Derivative 72)

0.5 g of the compound obtained in step (b) in solution in 50 ml of dryTHF are coupled with 0.4 g of glycine benzyl ester tosylate inaccordance with the Example 43c.

EXAMPLE 82 (a) 2-methylene 3-β-indolyl propanoic acid

(1) 2.61 g (0.01 mole) of 2-carboxy 3-β-indolyl propanoic acid ethylester obtained by the monosaponification of β-indolylmethyl malonicacid, itself obtained by the action of 2-chloromethyl indole on diethylmalonate according to Organic Synthesis, vol. 2, pp. 279, are stirred atroom temperature for 24 hours in accordance with Mannick (Ber 57, 1116(1924) with 2.23 g of 30% formol and 0.73 g of diethylamine. 5 ml ofwater are added and the organic phases are extracted with 3×50 ml ofether. The organic phases are washed in succession with 20 ml of 0.5NHCl, then with 20 ml of water and finally with 20 ml of NaCl saturatedsolution. The product is dried on Na₂ SO₄ and concentrated to dryness.

A pale yellow oil which crystallizes slowly is obtained.

M.P. 40°-60° C.; W 2.10 g; Rf 0.89 in CHCl₃ /EtOH (9/1); NMR correct.

(2) 2 g of the preceding ester are dissolved in 20 ml of ethanol andstirred with 1.2 equivalent of 1N NaOH solution for 20 hours at roomtemperature. The product is evaporated to dryness and taken up with 20ml of water, and extracted once with ether. The aqueous phase isacidified to pH 2 with 2N HCl. The material is extracted with 2×50 ml ofether, washed with water, dried on Na₂ SO₄ and evaporated to dryness.

After the usual successive treatments, 0.66 g of a brown oil isobtained.

Rf 0.57 in CHCl₃ /MeOH, 9/1. NMR correct

EXAMPLE 83 N-[3-(N-hydroxy formamido 2-β-indolylmethyl propanoyl]glycine (Derivative 73)

0.55 g of the compound 82 is hydrogenated at ordinary pressure under theconditions of Example 32. 0.42 g of a white solid is obtained.

M.P. 228° C.; Rf 0.38 in (A)

EXAMPLE 84 N-[3-(N-benzyloxy formamido) 2-isobutyl propanoyl]-glycinebenzyl ester (Derivative 74)

(a) 3.12 g (0.017 mole) of the isobutyl malonate ethyl monoesterobtained by the action of isobutyl chloride on diethyl malonate followedby a monosaponification, are stirred for 24 hours at room temperaturewith 1.5 g of 30% formol and 1.24 g of diethylamine. After treatment asin Example 82a-1, 2.8 g of an oil are obtained. Rf 0.92 in CHCl₃ /MeOH,9/1; NMR correct. The oil is employed crude for the following step.

(b) 2.5 g of the preceding ester are dissolved in 20 ml of ethanol andsaponified according to the conditions of Example 82a-2. 1.8 g of a paleyellow solid are obtained.

M.P. 65° C.; NMR correct, C, H, N corresponds to 2-isobutyl acrylicacid.

(c) 1.5 g of 2-isobutyl acrylic acid are condensed withO.benzylhydroxylamine in accordance with Example 82b-1. The oil obtainedhas a correct NMR spectrum.

W 1.6 g; Rf 0.18 in CHCl₃ /MeOH.

(d) 1.5 g of the preceding oil are added to 16 ml of HCO₂ H and themixture is treated as in Example 35. The oil obtained has a correct NMRspectrum and is employed for the following peptidic condensation.

W 1.35 g; Rf 0.31 in CHCl₃ /MeOH.

(e) 1.07 g of the preceding compound are coupled in solution in THF with1.20 g of glycine benzyl ester tosylate, and then the mixture is treatedas in Example 43c. 1.84 g of the compound 84 are obtained. The paleyellow oil is obtained pure by chromatography on a silica gel columnwith the use of the mixture CHCl₃ /MeOH, 8/2, as eluent.

NMR correct; C, H, N; RF 0.34 in CHCl₃ /MeOH, 9/1.

EXAMPLE 85 N-[3-(N-hydroxy formamido) 2-isobutyl propanoyl]-glycine(Derivative 75)

1.2 g of the preceding compound are hydrogenated at ordinary pressureunder the conditions of Example 32 which gives 0.62 g of a white solid.

M.P. 164°-170° C.; NMR correct; C, H, N.

EXAMPLE 86 (a) 2-N-benzyloxycarbamoyl 3-isopropyl propanoic acid

2 g (0.0106 mole) of isobutylmalonate ethyl monoester prepared accordingto Example 84a are coupled with 1.30 g O-benzylhydroxylamine base underthe conditions of the peptidic synthesis (Example 29). After the usualtreatment, a colourless oil is obtained which is saponified with 1N NaOHat 5° C. for 5 hours in ethanol. After acidification, extraction withether and evaporation to dryness a white solid is obtained.

W 2.1. g; M.P. 161° C. (EtOH); NMR correct; C, H, N.

(b) N-[2-(N-benzyloxycarbamoyl 3-isopropyl propanol] glycine benzylester (Derivative 76)

1.5 g of the preceding compound are coupled with 2.2 g of glycine benzylester tosylate according to the conditions of Example 33b. Aftertreatment, 1.2 g of a pale yellow solid are obtained.

M.P. 50°-60° C.; NMR correct; C, H, N.

EXAMPLE 87 N-[2-(N-hydroxycarbamoyl) 3-isopropyl propanoyl] glycine(Derivative 77)

1 g of the preceding compound is hydrogenated at oridinary pressure asin Example 32. 527 mg of a white solid are obtained.

M.P. 147°-151° C. (H₂ O); Rf 0.38 in (A); NMR correct; C, H, N.

EXAMPLE 88 ##STR32##

5 g of β-ethyl-α-benzalsuccinate are dissolved in 20 ml of isobutylene.After stirring for 24 hours at room temperature, either is added tothemixture. The organic solution is washed with 4% bicarbonate andwater, then dried on Na₂ SO₄ and exposed to dryness, which gives 4.96 gof an oil.

Rdt 80%; NMR correct; C, H, N; Rf 0.9 in CHCl₃ /CH₃ OH 9/1

EXAMPLE 89 ##STR33## α-benzalsuccinic acid monoterbutylate

4.5 g of the preceding compound are dissolved in 50 ml of ethanol. Thesolution is cooled to 0° C. and 34 ml of N sodium hydroxide. Afterstirring for 1 hour at 0° C. and one night at room temperature, themixture is evaporated under a vacuum and the residue is taken up with 30ml of water. The pH is brought to 1 by the addition of 2n HCl and theproduct is extracted with ether. The organic solution is washed withwater, dried on Na₂ SO₄ and evaporated under a vacuum. 4.07 g ofcream-coloured crystals are obtained.

Rdt 80%; M.P. 114° C.; Rf 0.51 in CHCl₃ /CH₃ OH 9/1 NMR correct; C, H,N.

EXAMPLE 90 ##STR34##

4 g of the preceding compound are reacted under the peptidic couplingconditions with 2.43 g of benzylhydroxylamine hydrochloride. After theusual treatments, 5.49 g of a thick oil are obtained.

Rdt 98%; Rf 0.52 in CHCl₃ /CH₃ OH 9/1. NMR correct; C, H, N.

EXAMPLE 91 ##STR35##

5 g of the preceding compound are stirred at 0° C. in the presence of 14ml of TFA for 3 hours. The product is evaporated under a vacuum. The oilobtained is triturated a plurality of times with dry ether, which gives3.4 g of a gummy solid.

Rdt 80%, Rf 0.8 in butanol/acetic acid/water 4.1.1. NMR correct; C, H,N.

EXAMPLE 92 ##STR36## Derivative 78

3.3 g of the preceding compound are put in the presence of 1.33 g ofglycine methyl ester hydrochloride under the peptidic couplingconditions. After the usual treatments, 3.53 g of a white powder areobtained.

Rdt 87%; W 102° C.; Rf 0.65 in CHCl₃ /CH₃ OH 9/1 NMR correct; C, H, N.

EXAMPLE 93 ##STR37## Derivative 79

3.5 g of the preceding compound are dissolved in 30 ml of 3/1 mixture ofmethanol and water. 11 ml of N sodium hydroxide are added to thesolution cooled to 0° C. After stirring for 1 hour at 0° C. and for 3hours at room temperature, the pH is brought to 1 with N HCl. Theproduct is extracted with ether. The organic solution is washed withwater, dried on Na₂ SO₄ and evaporated under a vacuum. The powerobtained is crystallized in ether to give 8.75 g of white crystals.

Rdt 82%; M.P. 171° C.; Rf 0.83 in butanol acetic acid/water 4.1.1. NMRcorrect; C, H, N.

EXAMPLE 94 ##STR38## Derivative 80

0.35 g of the preceding compound are dissolved in methanol and stirredat ordinary pressure in the presence of palladium on charcoal for 3hours. After having filtered the catalyst, the solvent is evaporatedunder a vacuum to give 0.24 g of a thick oil.

Rdt 90%; Rf 0.54 in butanol/acetic acid/water 4.1.1. NMR correct; C, H,N.

EXAMPLE 95 ##STR39##

300 mg of the compound of Example 93 are put in the presence of 72 mg of2-dimethylamino-ethane under the peptidic coupling conditions. After theusual treatments, 317 mg of an oil are obtained.

Rf 0.55 in CHCl₃ /Ch₃ OH 9/1.

Rdt 89%; NMR correct; C, H, N.

EXAMPLE 96 ##STR40## Derivative 82

310 mg of the preceding compound are hydrogenated under the conditionsof the compound 7 so as to give a pasty solid which crystallizes inmethanol in the form of white crystals.

W 192 mg; Rdt 80%; Rf 0.42 in CHCl₃ /CH₃ OH/CH₃ COOH 9/1.0.5--NMRcorrect; C, H, N.

EXAMPLE 97 ##STR41## Derivative 83

100 mg of the preceding compound are dissolved in 0.5 ml ofacetonitrile. There is added to the solution 0.05 ml of an aqueoussolution of 30% of H₂ O₂ at 0° C. After stirring for 24 hours at roomtemperature, 10 ml of butanol and a 5% aqueous solution of NaCl areadded. The organic solution is washed with water, and then evaporatedunder a vacuum to give 84 mg of a thick oil.

Rdt 80%; Rf 0.13 in butanol/acetic acid/water 4/1/1. NMR correct; C, H,N.

EXAMPLE 98 ##STR42## Derivative 84

0.5 g of the compound are put in the presence of 368 mg ofbenzyl-hydroxyproline methyl ester hydrochloride under the peptidiccoupling conditions. After the usual treatments, 515 mg of an oil areobtained.

Rdt 90%; RF 0.33 in CHCl₃ /H₃ COH 9/1; NMR correct; C, H, N.

EXAMPLE 99 ##STR43## Derivative 85

500 mg of the preceding compound are saponified under the conditions ofthe compound No. 6 so as to give 405 mg of a foam.

Rdt 83%; Rf 0.53 in CHCl₃ /CH₃ OH 7/3; NMR correct; C, H, N.

EXAMPLE 100 ##STR44## Derivative 86

400 mg of the preceding compound are hydrogenated under the conditionsof the compound of Example 94 to give 252 mg of a resinous product.

Rdt 92%, Rf 0.31 in CHCl₃ /CH₃ OH/CH₃ COOH 9/1/0.5 NMR correct; C, H, N.

EXAMPLE 101 ##STR45## Derivative 87

350 mg of the compound of Example 93 are combined with 128 mg ofmethionilol under the conditions of peptidic coupling to give 401 mg ofan oil.

Rdt 87%; Rf 0.28 in CHCl₃ /CH₃ OH 7/3; NMR correct; C, H, N.

EXAMPLE 102 ##STR46## Derivative 88

350 mg of the preceding compound are hydrogenated under the conditionsof the compound of Example 94 to give 257 mg of an oil.

Rdt 90%; Rf 0.30 in butanol/acetic acid/water 4/1/1. NMR correct; C, H,N.

EXAMPLE 103 ##STR47## Derivative 89

150 mg of the preceding compound are treated under the conditions of thecompound of Example 96 to give 156 mg of a thick oil.

Rdt 75%; Rf 0.20 in CHCl₃ /CH₃ OH 7/3. NMR correct; C, H, N.

EXAMPLE 104 ##STR48##

1 g of β-ethyl α-benzalsuccinate is combined with 715 mg of glycinemethyl ester hydrochloride under peptidic coupling conditions to give1.48 g of an oil.

Rdt 100%; Rf 0.82 in CHCl₃ /CH₃ OH 9/1; NMR correct; C, H, N.

EXAMPLE 105 ##STR49##

1.4 g of the preceding compound are subjected to an alkaline hydrolysisunder the conditions of the compound of Example 93 to give 1.06 g of athick oil.

Rdt 83%; Rf 0.77 in butanol/acetic acid/eau 4/1/1. NMR correct; C, H, N.

EXAMPLE 106 ##STR50## Derivative 90

1 g of the preceding compound is combined with 802 mg ofN-methylobenzylhydroxylamine trifluoroacetate under the peptidiccoupling conditions to give an oil which, in crystallizing, yields 984mg of white crystals.

Rdt 70%; M.P. 125° C.; Rf 0.69 in CHCl₃ /CH₃ OH 9/1; NMR correct; C, H,N.

EXAMPLE 107 ##STR51## Derivative 91

900 mg of the preceding compound are dissolved under the conditions ofthe compound of Example 94 to give 651 mg of an oily product.

Rdt 91%; Rf 0.59 in CHCl₃ /CH₃ OH 9/1; NMR correct; C, H, N.

EXAMPLE 108 ##STR52## Derivative 92

600 mg of the preceding compound are hydrolyzed under the conditions ofthe compound of Example 91 to give 393 mg of a thick oil.

Rdt 98%; Rf 0.22 in CHCl₃ /CH₃ OH/CH₃ COOH 9/1/0.5 NMR correct; C, H, N.

EXAMPLE 109 ##STR53## Derivative 93

500 mg of the compound of Example 104 are combined with 117 mg ofo-acetyl-hydroxylamine under the peptidic coupling conditions to give526 mg of a thick oil.

Rdt 89%; Rf 0.65 in CHCl₃.CH₃ OH 9/1; NMR correct; C, H, N.

EXAMPLE 110 ##STR54## Derivative 94

500 mg of the preceding compound are hydrolyzed under the conditions ofthe compound No. 4 to give 332 mg of pasty product.

Rdt 78%; Rf 0.63 in butanol/acetic acid/water 4/1/1. NMR correct; C, H,N.

EXAMPLE 111 ##STR55## Derivative 95

300 mg of the preceding compound are hydrogenated under the conditionsof compound No. 7, which gives 280 mg of an oily product.

Rdt 93%; Rf 0.60 in butanol/acetic acid/water 4/1/1; NMR correct; C, H,N.

EXAMPLE 112 ##STR56##

500 mg of the compound of Example 104 are combined with 190 mg ofo-benzyl-hydroxylamine under the conditions of peptidic coupling, whichgives 564 mg of a thick oil.

Rdt 85%; Rf 0.54 in CHCl₃ /CH₃ OH 9/1. NMR correct; C, H, N.

EXAMPLE 113 ##STR57## Derivative 96

500 mg of the preceding compound are treated with trifluoroacetic acidunder the conditions of compound No. 4 to give 349 mg of a foamyproduct.

Rdt 77%; Rf 0.6 in butanol/acetic acid/water 4/1/1. NMR correct; C, H,N.

EXAMPLE 114 ##STR58## Derivative 97

300 mg of the preceding compound are hydrogenated under the conditionsof the compound No. 94 to give 268 mg of an oily product.

Rdt 89%; Rf 0.55 in butanol/acetic acid/eau 4/1/1.

EXAMPLE 115 ##STR59##

366 mg of the compound of Example 89 are combined with 471 mg of glycinemethyl ester paratoxylate under the peptidic coupling conditions, whichgives 570 mg of a fluid oil.

Rdt 100%; Rf 0.83 in CHCl₃ /CH₃ OH 9/1; NMR correct; C, H, N.

EXAMPLE 116 ##STR60##

500 mg of the preceding compound are treated with trifluoroacetic acidunder the conditions of the compound of Example 91 to give 302 mg of awhite powder.

M.P. 145° C.; Rdt 70%; Rf 0.16 in CHCl₃ /CH₃ OH 9/1 NMR correct; C, H,N.

EXAMPLE 117 ##STR61## Derivative 98

300 mg of the preceding compound are combined with 150 mg ofo-benzyl-hydroxylamine hydrochloride under the peptidic couplingconditions to give 81 mg of white crystals.

M.P. 151° C.; Rdt 60%; Rf 0.5 in CHCl₃ /CH₃ OH 9/1. NMR correct; C, H,N.

EXAMPLE 118 ##STR62##

78 mg of the preceding compound are hydrogenated under the conditions ofthe compound of Example 94 to give 46 mg of a white product.

M.P. 129° C.; Rdt 97%; Rf 0.38 in butanol/acetic acid/water 4/1/1. NMRcorrect; C, H, N.

EXAMPLE 119 N-(N-benzyloxy-3-carbamoyl-2-benzylidene)glycine piperidinoethyl ester Derivative 100

3 g of the compound of Example 91 are combined with 2.5 g of glycinepiperidino ethyl ester trifluoroacetate under the conditions of peptidicsynthesis. After the usual treatments, 3.5 g of a white powder areobtained.

M.P. 106° C.; Rdt 65%; Rf 0.62 in CHCl₃ /CH₃ OH 9/1 NMR correct; C, H,N.

Example 120 ##STR63## N-(N-3-hydroxycarbamoyl-2-benzyl-propanoyl)glycinepiperidino ethyl ester Derivative 101

1 g of the preceding compound is hydrogenated at ordinary pressure underthe conditions of Example 32. 0.76 g of a crystallized white solid isobtained.

M.P. 231° C.; Rdt 100%; Rf 0.7 in (A); NMR correct; C, H, N.

The results of the biological and pharmacological studies reported belowshow the interesting enkephalinase-inhibiting, antalgic, antidepressive,antidiarrhea and hypotensive properties of the derivatives of formula I.

Consequently, an object of the present invention is to provide apharmaceutical composition having in particularenkephalinase-inhibiting, antalgic, antidepressive, andidiarrhe andhypotensive properties, said composition comprising, as an activeingredient, a compound of formula I or an addition salt with apharmaceutically acceptable acid or base of said compound.

A--BIOLOGICAL STUDY (I) Dosage of the "enkephalinasic" activity(enkephalin dipeptidylcarboxypeptidasic) and determination of the effectof the inhibitors

The enzyme preparation employed is a membrane fraction from the striatumof the rat or mouse.

This fraction is obtained by homogenization at 4° C. in 20 volumes ofTris-Hl buffer 0.05M (pH 7.4) followed by two successive centrifugations(1000 g×min. and 200,000 g×min.) at the end of which the sediment of thesecond centrifugation is retained. It is washed by resuspension in 10 mlof the buffer followed by centrifugation (200,000 g×min.) and theresulting sediment is in turn washed superficially to complete theremoval soluble enzymes. The resulting membrane fraction is taken upinto the buffer at 4° C. to obtain a suspension comprising about 1.5 mgof proteins per ml.

An aliquot sample (50 μl) of the membrane suspension is then incubatedin a final volume of 100 μl at 25° C. in the presence of 10 nMleucine-enkephalin ³ H (39 Ci/mmole), previously purified by columnchromatography over Porapak Q(100-120 mesh, Waters Assoc.) and 0.1 mMpuromycine, an aminopeptidase inhibitor. The incubation time isgenerally set at 15 min. so as to determine the initial rate offormation of tripeptide Tyr-Gly-Gly-³ H caracteristic of the enkephalindipeptidylcarboxypeptidasic (enkephalinasic) activity. The reaction isterminated by the addition of 25 μl of 0.2N HCl and the tripeptide isisolated by column chromatography over Polopak Q or on a thin layer ofsilica, according to the methods described by Malfroy et al (B. Malfroy,J. P. Swerts, C. Llorens and J. C. Schwartz, Neuro-Science Letters, 11,329, 1979).

The results obtained with either method have always been consistent.

Determination of the radioactivity of the tripeptide is effected byliquid scintillation spectrometry.

The effect of the inhibitors is established by experiments withincreasing concentrations of such materials, which leads to thedetermination of the 50% inhibitory concentrations calculated by meansof data according to the method of Parker and Waud (J. Pharmacol. Exper.Ther. 177, 1, 1971). In some cases, the competitive nature of theinhibition was established by incubation experiments with a fixedconcentration of the inhibitor and increasing concentrations of thesubstrate.

    ______________________________________                                        Derivative No.                                                                             50% inhibitory concentration                                     ______________________________________                                         2           7 × 10.sup.-8 M                                             4           1 × 10.sup.-7 M                                            28           8 × 10.sup.-9 M                                            30           1 × 10.sup.-7 M                                            38           3 × 10.sup.-9 M                                            40           5 × 10.sup.-9 M                                            47           3 × 10.sup.-9 M                                            63           5 × 10.sup.-8 M                                            69           7 × 10.sup.-9 M                                            71           2 × 10.sup.-7 M                                            99           3 × 10.sup.-9 M                                            ______________________________________                                    

(II) Protection of the endogenic enkephalinases liberated bydepolarisation of the brain sections

Stratium sections of the rat are incubated in a medium of Krebs-Ringerand the (Met⁵) enkephalin liberated by addition of 50 mM KCl isevaluated by radioimmunologic dosage (Patey and Coll., Science, 1981,212, 1153-1155). In the presence of thiorpan, an enkephalinaseinhibitor, there is observed an increased recovery of the pentapeptide,which is approximately doubled.

In the presence of a plurality of the compounds according to the presentinvention, a significantly higher recovery is observed relative to thatobserved under the effect of thiorphan, even when the latter is presentin supramaximal concentration. The higher protection of the liberatedenkephalins afforded by these compounds is thence attributed to theinhibition of peptidases (probably aminopeptidases) other than thosewhich are sensitive to thiorphan.

                  TABLE I                                                         ______________________________________                                        Protection of the liberated endogenic                                         enkephalins by depolarization of strium                                       sections of the rat                                                                            (Met.sup.5) liberated enkephalin                             Added inhibitor  (pmole/g/min.)                                               ______________________________________                                        None             120 ± 25                                                  Thiorphan (10.sup.-7 M)                                                                        280 ± 30*                                                 Derivative No. 28 (10.sup.-6 M)                                                                550 ± 40*                                                 Derivative No. 38 (10.sup.-7 M)                                                                600 ± 30*                                                 Derivative No. 47 (10.sup.-7 M)                                                                630 ± 50*                                                 Derivative No. 99 (10.sup.-7 M)                                                                600 ± 25*                                                 ______________________________________                                         *p < 0.001                                                                    ##STR64##                                                                

B--PHARMACOLOGICAL STUDIES

The pharmacological study of the products described above showed aspecific antalgic, antidiarrhea, antidepressive and hypotensive effectand a potentiation action of the effects of an enkephalin, D Ala₂ MetEnkephalin (in particular antalgic and hypotensive).

The following pharmacological tests were conducted:

(I) Acute Toxicity

The determination of the death rate in the mouse is observed after asingle intravenous administration of increasing doses of the testcompounds.

For all the compounds tested, the LD₅₀ is in excess of 100 mg/kg/i.v.and 400 mg/kg by the intraperitoneal route.

(II) Subacute Toxicity

The derivative 63 was administered by the intraperitoneal route forthree weeks to mice at the dose of 50 mg/kg three times a day (150mg/kg/day). The animals did not exhibit any change in the weightincrease rate or any sign of toxicity with respect to the controls. Theweight of the organs and their anatomic-pathologic examination aftersacrificing the animals showed no difference with respect to the solventcontrols.

Further, in the animals in which a reversability test was conducted upontermination of the treatment, no sign of tolerance or habit forming andno weaning phenomena were observed.

(III) Antalgic Activity (1) Hot plate test

Licking reflex of mice on a plate heated at 55° C. according to themethod of Jacob et co-workers (Arch. Int. Pharmacodyn. 122, 287-300,1959: 133, 296-300, 1961).

(a) Potentiation of the antalgic effect of DAla₂ Met Enkephalin:

(α) By the intraventricular route (ivt):

The table I shows that the effect of a subactive dosage (0.3γ) ofDAla_(A) Met Enkephalin when administered by the intraventricular routeis significantly potentiated (p<0.05) by the derivatives 28 and 38 atthe dose of 30γ and that this effect is antagonised by Naloxone.

(β) By the intravenous route:

When administered by the intravenous route, the derivatives No. 2, 30and 63 at the dose of 30 mg/kg produce a 100% increase in the lickingtime with respect to animals treated with DAla₂ Met Enkephalin (0.3γintraventricular).

                  TABLE I                                                         ______________________________________                                        Hot plate                                                                     Potentiation of the antalgic effect of                                        D Ala.sub.2 Met Enkephalin and Naxolone-induced                               antagonism                                                                                                  % increase in the                                                             licking time with                                              Dose           respect to animals                                             γ/                                                                              No     treated with D                                                 mouse   of     Ala.sub.2 Met enkephalin                        Derivative     ivt     mice   (1)                                             ______________________________________                                        n° 28 + 30      6      350*                                            DAla.sub.2 Met Enkephalin (1)                                                 n° 38 + 30      6      320*                                            DAla.sub.2 Met Enkephalin (1)                                                 n° 28 + 30      10     18                                              DAla.sub.2 Met Enkephalin +                                                   Naloxone (10 mg/kg ss.                                                        cut.) (1)                                                                     n° 47   30      10     300*                                            n° 99   30      10     280*                                            ______________________________________                                         (1) the dose of D Ala.sub.2 Met Enkephalin is 0.3 γ/mouse, which        dose is inactive in itself                                                    *P < 0.05 Wilcoxon's level test                                          

(b) Specific antalgic effect

By the intravenous route.

The following Table shows that the derivatives No. 2, 30 and 63 have anantalgic effect in the hot plate test (55° C.).

    ______________________________________                                        Derivative   ED.sub.50 mg/KG/i.v.                                             ______________________________________                                         2           30                                                               30           18                                                               63            3                                                               47           25                                                               99           30                                                               ______________________________________                                    

(2) Test in mice of the withdrawal of the tail immersed in water heatedto 48° C. according to the method of Sewell and Spencer(Neuropharmacology, 1976, 15, p. 683-688).

The derivatives 2 and 30 administered by the intravenous route at thedose of 30 mg/kg potentiate very significantly the DAla₂ Met Enkephalinadministered 15 minutes later by the intraventricular route, at varyingsubactive doses of from 10 to 30γ/mouse.

This effect is durable and exceeds 2 hours; it is also observed at thedose of 5 mg/kg with the derivative No. 63.

(3) Phenylbenzoquinone or "writhing" test according to the method ofSiegmund and co-workers (Proc. Soc. Expert Biol. Med. 1957, 95,729-731).

The derivatives 2, 30 and 63 injected respectively at the dose of 15, 18and 1 mg/kg i.v., protect the treated animals fromphenylbenzoquinone-induced pain with a significant difference withrespect to the controls p<0.01. This effect is antagonized by Naloxone.

Here again, if the antinociceptive effects of these compounds arecompared with those of Thiorphan, a selective enkephalinase-inhibitor, adistinctly greater maximum effect is observed. For example, thiorphan,even at a heavy dose, never completely eliminates the abdominalstretchings of the mouse, whereas a complete elimination thereof isobserved in the case of these compounds. This more pronounced analgesiceffect is attributable to an improved protection of the endogenic opioidpeptides, in relation with the inhibition not only of the enkephalinase,but also of the aminopeptidases involved in their hydrolysis.

(IV) Antidepressive Activity

Forced swimming or "Mice Despair" test according to Porsolt's method.

This test is representative of an antidepressive effect.

At the dose of 1 mg/kg i.v., the derivative No. 63 significantly reduces(p<0.001) the duration (in seconds) of the immobile periods with respectto the controls.

(V) Antidiarrhea Activity

It was studied according to the method of Niemegeers and co-workers(Arzneim, Forsh 22, 516, 1972) in which the diarrhea is produced in therat by means of castor oil.

This effect was moreover found in respect of the doses of 10-30 mg/kgand is antagonized by Naloxone.

(VI) Hypotensive Activity

Specific enkephalin-potentiating activity, (Laubie M. Schmith H.,Vincent M., Remon D. Central cardiovascular effects of Morphinometicpeptides in dogs. European Journal of Pharmacology, vol. 46, 67-71,1977).

By the intravenous route, the derivative No. 63 resulted in a reductionin the blood pressure at the dose of 10 mg/kg.

The results of these studies demonstrate the low toxicity and theinteresting enkephalinase-inhibiting, antalgic, antidepressive,antidiarrhea and hypotensive properties of the derivatives of theinvention which make them applicable in human and veterinary medicine.

The pharmaceutical composition of the invention may be administered tohumans by the oral, parenteral or rectal route.

Each unit dose advantageously contains 0.5 to 100 mg of activeingredient. The daily doses administrable may vary from 0.5 mg to 1200mg of active ingredient.

Having now described our invention what we claim as new and desire tosecure by Letters Patent is:
 1. An amino acid derivative of the formula##STR65## wherein R₁ is phenyl optionally mono- or polysubstituted withhalogen;R₂ is hydrogen or methyl; Z is --(CH₂)-- or ═(CH)--; p is 0 whenZ is ═(CH)-- and 1 when Z is --(CH₂); R₃ is hydroxy; C₁₋₄ alkoxyoptionally mono-substituted with halogen; phenyl C₁₋₄ alkoxy whosephenyl group is optionally substituted with halogen; C₁₋₆ alkyl amino;phenyl C₁₋₄ alkyl amino whose phenyl group is optionallymono-substituted with halogen; X is disubstituted aminomethyl of theformula ##STR66## in which R" is hydrogen, benzyl, benzoyl or C₁₋₄alkoxy carbonyl and R"' is hydrogen, methyl, benzyl, fluorobenzyl ortrifluoroethyl.
 2. A compound according to claim 1, selected from##STR67##
 3. An amino acid derivative of the formula ##STR68## whereinR₁ is phenyl optionally mono-substituted with halogen;R₂ is hydrogen ormethyl; Z is --(CH₂)-- or ═(CH)--; p is 0 when Z is ═(CH)-- and 1 when Zis --(CH₂); R₃ is hydroxy; C₁₋₄ alkoxy optionally mono-substituted withhalogen; C₁₋₄ alkoxy substituted with dialkylamino in which the alkylgroups contain 1-4 carbon atoms; phenyl C₁₋₄ alkoxy whose phenyl groupis optionally substituted with halogen; C₁₋₆ alkyl amino; phenyl C₁₋₄alkyl amino whose phenyl group is optionally mono-substituted withhalogen; X is monosubstituted carbamoylmethyl of the formula --CH₂--CO--NH--OR" in which R" is benzyl, benzoyl or C₁₋₄ alkoxy carbonyl. 4.A compound according to claim 3 selected from: ##STR69##
 5. An aminoacid derivative of the formula ##STR70## wherein R₁ is hydrogen; C₁₋₄alkyl; phenyl optionally mono-substituted with halogen; or indolyl;R₂ ishydrogen; methyl; or alkyl optionally substituted with indolyl; Z is--(CH₂)-- or ═(CH)--; p is 0 when Z is ═(CH)-- and 1 when Z is--(CH₂)--; R₃ is hydroxy; C₁₋₄ alkoxy optionally mono-substituted withhalogen; phenyl C₁₋₄ alkoxy whose phenyl group is optionally substitutedwith halogen; C₁₋₆ alkyl amino; or phenyl C₁₋₄ alkyl amino whose phenylgroup is optionally mono-substituted with halogen; X is disubstitutedaminomethyl of the formula ##STR71## or a mono- or disubstitutedcarbamoylmethyl of the formula ##STR72## in which R" is hydrogen,benzyl, benzoyl or C₁₋₄ alkoxy carbonyl, R"' is hydrogen, methyl, benzylfluorobenzyl or trifluoroethyl, and R₄ is benzyl, benzoyl or C₁₋₄ alkoxycarbonyl.
 6. An amino acid derivative according to claim 5, wherein X isa disubstituted aminomethyl of the formula ##STR73## in which R" ishydrogen, benzyl, benzoyl, or C₁₋₄ alkoxy carbonyl and R"' is hydrogen,methyl, benzyl, fluorobenzyl or trifluoroethyl.
 7. A compound accordingto claim 6, selected from the following: ##STR74##
 8. An amino acidderivative of the formula ##STR75##